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Angelique Nicole Besold
Graduate Research Assistant
University of Maryland, School of Pharmacy
Department of Pharmaceutical Sciences
20 Penn Street HSFII Room 512
Baltimore, MD 21201
410-706-3487
[email protected]
Research Interest
My research focuses on two zinc finger (ZF) proteins that are involved in neuronal
development: Neural Zinc Finger Factor-1 (NZF-1) and Myelin Transcription Factor 1
(MyT1). These proteins belong to a class of ZFs that contains a unique CCHHC motif in
each ZF domain. These domains contain a high degree of sequence similarity, yet these
proteins recognize and regulate different genes. Their DNA recognition properties are not
well understood, though it is of great interest as both proteins have been implicated in a
number of neurological disorders. By using a series of biophysical and biochemical
techniques, I am interested in understanding the relationship between metal coordination,
protein folding, and DNA recognition.
Education
Ph.D in Pharmaceutical Sciences., Department of Pharmaceutical Sciences, University of
Maryland at Baltimore School of Pharmacy, 2009 – 2014, Advisor: Dr. Sarah L.J.
Michel
B.A. in Biology, College of Notre Dame of Maryland, Baltimore, MD, 2004 – 2008, Cum
Laude
Honors, Awards, and Affiliations
 Graduate Student of the Month – University of Maryland School of Pharmacy,
September 2014
 National Institute of Neurological Disorders and Stroke Predoctoral Fellowship
F31NS074768, July 01, 2011 – June 30, 2014
 Graduate Student of the Month – University of Maryland School of Pharmacy,
February 2014
 Pharmaceutical Sciences Graduate Merit Award, September 2013
 Robert G. Pinco Endowed Scholarship – Merit Award, September 2012
 Society of Biological Inorganic Chemistry Student Travel Grant, May 2011
 Society of Biological Inorganic Chemistry Honorable Mention Poster Award,
August 2011
 Graduate Student of the Month – University of Maryland School of Pharmacy,
February 2010
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Member, Beta Beta Beta – National Biological Honor Society
Member, Delta Epsilon Sigma – National Scholastic Honor Society
Member, Rho Chi – Academic Honor Society in Pharmacy
Member, American Chemical Society
Member, Society of Biological Inorganic Chemistry
Publications
Besold, A.N.; Widger, L.R.; Namuswe, F.; Michalek, J.L.; Michel, S.L.J.; Goldberg, D.P.
2014. A Designed Zinc Finger Peptide with Hydrolytic Activity. In preparation
Besold, A.N and Michel. 2014. The Neural Zinc Finger Factor/Myelin Transcription
factor proteins: a unique family of “non-classical” zinc finger proteins. Submitted to
Biochemistry
Besold, A.N.; Amick, D.L.; Michel, S.L.J. 2014. A role for hydrogen bonding in DNA
recognition by the non-classical CCHHC type zinc finger, NZF-1. Mol. Biosyst.,
10(7):1753-6. PMID: 24820620
Gilbreath, J.J.; Pich, O.Q.; Benoit, S.L.; Besold, A.N.; Maier, R.J.; Michel, S.L.J.;
Maynard, E.L.; Merrel, D.S. 2013. Random and Site-Specific Mutagenesis of the
Helicobacter pylori Ferric Uptake Regulator Provides Insight into Fur Structure-Function
Relationships. Mol. Micro., 89(2):304-23. PMID: 23710935
Besold, A.N.; Oluyadi, A.A.; Michel, S.L.J. 2013. Switching Metal Ion Coordination and
DNA Recognition in a Tandem CCHHC-type Zinc Finger Peptide. Inorg. Chem,
52(8):4721-8. PMID: 23521535
Michalek, J.L.; Besold, A.N; Michel, S.L.J. 2011. Cysteine and histidine shuffling:
mixing and matching cysteine and histidine residues in zinc finger proteins to afford
different folds and function. Dalton Trans, 40(47):12619-32. PMID: 21952363
Lee, S.J.; Michalek, J.L.; Besold, A.N.; Rokita, S.; Michel, S.L.J. 2011. Classical
Cys2His2 zinc finger peptides are rapidly oxidized by either H2O2 or O2 irrespective of
metal coordination. Inorg. Chem, 50(12):5442-50. PMDID: 21574551
Besold, A.N.; Lee, S.J.; Sue Lue, N.; Cymet, H.J. ; Michel, S.L.J. 2010. Functional
characterization of iron-substituted neural zinc finger factor 1: metal and DNA binding. J.
Biol. Inorg. Chem, 15(4):583-90. PMID: 20229093
Poster Presentations
Besold, A.N.; Oluyadi, A.A..; Amick, D.L.; Michel, S.L.J. Defining the Paradigm of DNA
Recognition by the Neural Zinc Finger Factor Family of Proteins. FASEB Trace Elements in
Biology and Medicine, Steamboat Spring, CO. June 2014.
Besold, A.N.; Oluyadi, A.A..; Amick, D.L.; Michel, S.L.J. Controlling DNA Recognition in the
Highly Homologous Neural Zinc Finger Factor Family of Proteins. Frontiers at the Chemistry
Biology Interface 7th Annual Symposium, University of Maryland, Baltimore, MD. May 2014.
Besold, A.N.; Sue Lue, N.; Cymet, H.J.; Michel, S.L.J. Characterization of metal coordinating
ligands of NZF-1, a unique zinc finger protein with a CCHHC ligand set. Mid-Atlantic
Seaboard Inorganic Symposium-4, The Johns Hopkins University, Baltimore, MD.
August 2012.
Besold, A.N.; Sue Lue, N.; Cymet, H.J.; Michel, S.L.J. Differential DNA binding properties of
two highly homologous zinc finger proteins: NZF-1 vs MyT1. International Conference of
Biological Inorganic Chemistry 15, University of British Columbia, Vancouver, B.C., Canada
V6T 1Z4. August 2011.
Besold, A.N.; Lee, S.J.; Sue Lue, N.; Cymet, H.J. ; Michel, S.L.J. Functional characterization of
iron substituted neural zinc finger factor-1. Frontiers at the Chemistry Biology Interface 3rd
Annual Symposium, The Johns Hopkins University, Baltimore, MD. May 2010.
Besold, A.N.; Lee, S.J.; Sue Lue, N.; Cymet, H.J. ; Michel, S.L.J. Functional characterization of
iron-substituted neural zinc finger factor-1. 32nd Annual Graduate Research Conference,
University of Maryland, Baltimore, Baltimore, MD; April 2010
Besold, A.N.; Lee, S.J.; Sue Lue, N.; Cymet, H.J.; Michel, S.L.J. Iron substituted neural zinc
finger factor-1. Frontiers at the Chemistry Biology Interface 2nd Annual Symposium, University
of Maryland, Baltimore County, Baltimore, MD; May 2009.
Teaching Experience
Mentor, Summer Interns: Trained and supervised two pharmacy students from University
of Maryland PharmD program as well as an undergraduate student from Morgan State
University. Students were trained on a variety of subjects including protein expression,
protein purification, UV-visible spectroscopy, and fluorescence anisotropy.
 Nia Ebrahim, Bryn Mawr High School: June 2014 – August 2014
 Deborah Amick, Elkton High School Chemistry Teacher: June 2013 – August
2013
 Abdulafeez Oluyadi, University of Maryland School of Pharmacy: September
2012 – January 2013
 Ting Liu, University of Maryland School of Pharmacy: September 2011 – May
2012
 Tiffany Strickland, Morgan State University: June 2009 – August 2009
Mentor, Spring into Maryland Science (SIMSI): A hands on “mini-graduate” school
experience for undergraduate women from the College of Notre Dame of
Maryland to graduate school. March 2011, March 2012, March 2013.
Head Teaching Assistant, Responsible for directing, proctoring and grading of exams as
well maintaining an interactive “Blackboard” site for a variety of courses.
 Integrated Science and Therapy III/IV: January 2011 – May 2011
 Biochemistry: August 2009 – December 2009, August 2010 – December 2010
 Microbiology and Antibiotics II: January 2010 – May 2010
 Pharmacokinetics: August 2009 – December 2009
Educator, Maryland Science Center: Educated children and adult on a variety of
subjects ranging from physics to chemistry to dinosaurs. May 2006 – May 2008
Volunteer Experience
 Back on My Feet, Non-resident member (volunteer). This non-profit organization
uses running as a means to promote self-sufficiency and build confidence among
those affected by homelessness in various shelters throughout Baltimore city. I
specifically run with homeless veterans from the Maryland Center for Veterans
Education and Training (MVET). June 2013 – April 2014.
Title of Dissertation: New Vistas in Zinc Finger Biochemistry: Examining the MetalMediated DNA Recognition of the Neural Zinc Finger Factor/Myelin Transcription
Factor Family of Non-Classical Zinc Finger Proteins and Creating Catalytic Moieties
from Zinc Finger Scaffolds
Angelique N. Besold, Doctor of Philosophy, 2014
Dissertation Directed by: Sarah L. J. Michel, Ph.D, Associate Professor, Department of
Pharmaceutical Sciences
The Neural Zinc Finger Factor/Myelin Transcription Factor (NZF/MyT) family of
‘non-classical’ zinc fingers (ZFs) is involved in regulating key genes during neuronal
development. These proteins contain domains with a CCHHC motif and utilize a CCHC
ligand set to coordinate zinc ions in a tetrahedral geometry and adopt secondary structure.
To better understand the metal-mediated DNA recognition properties of two members of
this family, NZF-1 and MyT1, we have taken a biochemical/biophysical approach
involving UV-visible spectroscopy, circular dichroism, and fluorescence anisotropy. Our
first efforts focused on the role of metal ion identity in function and utilized a two domain
construct of NZF-1 comprised of ZFs two and three (NZF-1-F2F3), which bind an
AAGTT DNA sequence located in the promoter of the β-retinoic acid receptor gene
(βRAR). We discovered that iron(II) can coordinate to the ZF domains with no alteration
in the DNA binding properties when compared to the zinc bound form. We then utilized a
mutagenesis approach to determine the role of a conserved histidine that is not involved
in metal coordination. We determined that this residue is involved in stabilizing a
hydrogen bonding interaction important for NZF-1-F2F3/βRAR binding. Furthermore, we
demonstrated that a single arginine residue in ZF3 of NZF-1, which is absent in MyT1, is
required for the NZF-1-F2F3/βRAR DNA interaction. These results suggest that the few
non-conserved amino acids present in the ZF domains of this family drive sequencespecific DNA recognition.
In addition, research aimed at engineering ZF domains to have catalytic activity
has been pursued. In one effort, a prototype classical CCHH ZF domain called Consensus
Peptide-1 (CP-1) was mutated at one of the metal coordinating cysteines. This resulted in
an open coordination sphere at the zinc site, making it accessible for hydrolysis of
substrates. Using 4-nitrophenylacetate as a probe, we demonstrated that this construct can
promote hydrolysis. We also created a chimeric protein composed of the RNA binding
ZF tristetraprolin (TTP) and the RNA cleavage protein Ribonuclease4 (RNase4). This
Chimera, TTP2D-RNase4, catalytically cleaves target RNA in vitro. This is the first step
towards our long term goal of engineering TTP as a novel anti-inflammatory/anti-cancer
agent.
New Vistas in Zinc Finger Biochemistry: Examining the Metal-Mediated DNA
Recognition of the Neural Zinc Finger Factor/Myelin Transcription Factor Family of
Non-Classical Zinc Finger Proteins and Creating Catalytic Moieties from Zinc Finger
Scaffolds
by
Angelique N. Besold
Dissertation submitted to the Faculty of the Graduate School of the
University of Maryland, Baltimore in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy
2014
Acknowledgements
Firstly, I would like to thank my advisor, Dr. Sarah Michel, for all of the support
and guidance she offered even before I began graduate school. I first met Sarah in 2007
when I came to her lab as an intern from the College of Notre Dame of Maryland and had
absolutely no clue what I was doing. That summer changed my outlook on research and
my future. I would not be writing these acknowledgements today were it not for that
experience. Graciously, she took me under her wing as a laboratory technician after
college and has been with me ever since. With her help, I have achieved more during my
time in graduate school than I thought possible. She has always been supportive and
optimistic, even when I screwed something up in the lab…which I recently did. It is with
sincere gratitude that I thank her for all she has done.
I could not have made this far without the support of my wonderful committee
members. I have known Dr. Paul Shapiro for just as long as I have known Sarah and I
credit him helping to spark my research interest. Not only did he teach me practical skills
like cell culture way back in 2007, but he has continued to advise me ever since. Dr.
Angela Wilks has been like a co-advisor during these past years for which I am grateful.
Her incredibly helpful comments and suggestions in group meeting helped me to think
about my science in a different way. Not only was she there when I was having an issue
with research, but she is also essential to getting any number of broken instruments in the
department fixed ASAP! Dr. Maureen Kane brought many interesting aspects to my
research from examining how retinoic acid alters the function of proteins to actually
getting me to work with animal tissue, which I never thought I would do. I thank her for
all of her time and all of the helpful discussions we have had on this wide range of topics.
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Dr. Gerald Wilson graciously stepped in as my outside committee member, which I
greatly appreciate. I am thankful to be part of an incredibly interesting project that he and
Sarah have started and I look forward to seeing how it turns out! Overall, I could not have
had a better committee and am so lucky to have gotten the chance to work with such
amazing scientists!
I am indebted to many people who helped me to begin this scientific journey
during my undergraduate years. It was my organic chemistry professor, Dr. Angela
Sherman, who encouraged me to pursue an internship in research despite the fact that I
was convinced it was absolutely the last thing I wanted to do in life (ha!). I would also
like to thank two of my incredible biology professors: Dr. Peter Hoffman and Dr. Cynthia
Wang. Not only were they by my side throughout college, but both have continued to
give me advice and support long after. They have gone above and beyond the call of
duty, for which I am forever grateful.
I am also very thankful to the Michel Lab members who I have had the pleasure
of working with: Dr. Seung Jae Lee, Dr. Ronny Dosanjh, Dr. Abby West, Dr. Sarah
Evans, Dr. Jamie Michalek, Geoffrey Shimberg, and Mohsin Khan. I would like to give a
special thanks to SJ, who taught me the basics in lab and brought the lab to life with his
singing (and rapping!). I am also eternally grateful to Jamie, who has become one of the
dearest friends that I have. She was and is always there for me through the good times
and the bad. She was there when I needed a good laugh or a shoulder to cry on. This past
year has not been quite the same without her in lab, but she has been just as supportive as
ever and I am so grateful for everything that she has done for me.
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I would like to thank the many members of our department who have been kind
and helpful to me on so many different levels. Dr. Ramin Samadani has gone beyond
what was needed in helping me with cell culture and western blots and he was always
there when I needed a laugh. The lab of Dr. Hongbing Wang has been incredibly helpful
the past couple of months while I learned how to do luciferase assays for the first time. In
particular, I would like to thank Dr. Caitlin Lynch, Brandy Grazel, and William Hedrich.
A big thank you to the lab of Dr. Edwin Pozharski, particularly Dr. Franz St. John and
Dr. Javier Gonzalez for their help with basically everything. A thank you to Geoffrey
Heinzl for helping me to FINALLY understand catalytic zinc sites! I am grateful to all of
the members of our group meeting for their helpful insight and discussion, so thank you
members of the Wilks, Oglesby-Sherrouse, and Wintrode labs. Of course I need to thank
my classmate, friend, and coffee buddy Dr. Diana Vivian who has really helped me get
through these past few months!
I would also like to thank the many collaborators that I have had throughout the
years who have truly taught me so much about science. Thank you Dr. Holly Cymet, Dr.
David Goldberg, Dr. Leland Widgar, Dr. Alison McQuilken, Dr. Ananya Majumdar, Dr.
Kellie Hom, Dr. Dean Wilcox, and anyone else who I may be forgetting.
Finally, I would like to thank my family and my future family, that of my
wonderful fiancé, for their support throughout the years. I am incredibly appreciative for
all of their kind words and encouragement. A particularly special thank you goes out to
my wonderfully incredible fiancé, Jonathan Skarin. He has been my biggest supporter
and I do not know how I could have done this without him. Even when I was going crazy
towards the end he was always there to make me smile and COMPLETELY take over all
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of the household chores while I worked away. I feel so lucky to have him by my side and
I love him dearly. Last, but not least, I have to thank my forever adorable cats Dr. Charlie
Bucket Besold-Skarin PhD, DO, Esq and Dr. Astronaut Dinosaur Skarin-Besold DDS, PI.
Seeing them every day always brightened my mood and helped me get through!
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Table of Contents
Chapter 1. The Neural Zinc Finger Factor/Myelin Transcription factor proteins: a
unique family of “non-classical” zinc finger proteins..………………………..……...1
1.1 Introduction ........................................................................................................ 1
1.2 Biological Role .................................................................................................. 4
1.3 Regulation of CCHHC ZF Proteins ................................................................... 7
1.4 Disease States Linked to NZF/MyT Proteins .................................................... 8
1.5 Metal Ion Site: Coordinating Ligands.............................................................. 10
1.6 Affinities of Co(II) and Zn(II) for the CCHHC Domains of NZF-1/MyT1 .... 12
1.7 Role of Non-Coordinating Histidine in CCHHC Domains ............................. 13
1.8 Fold of the CCHHC ZF Domains .................................................................... 14
1.9 DNA Partners ................................................................................................... 15
1.10 Conclusion ..................................................................................................... 18
1.11 Thesis ............................................................................................................. 19
1.12 References ...................................................................................................... 21
Chapter 2. Functional Characterization of Iron-Substituted Neural Zinc Finger
Factor 1: Metal and DNA Binding Affinities ............................................................... 37
2.1 Introduction ...................................................................................................... 37
2.2 Materials and methods ..................................................................................... 41
2.2.1 Preparation of NZF-1-F2F3 ...................................................................... 41
2.2.2 Metal-Binding Titrations........................................................................... 41
2.2.3 Co(II) and Zn(II) binding .......................................................................... 42
2.2.4 Fe(II) binding ............................................................................................ 42
2.2.5 Displacement of Fe(II) by Co(II) ............................................................. 43
2.2.6 Air oxidation of Fe(II)-NZF-1-F2F3......................................................... 43
2.2.7 Oligonucleotide probes ............................................................................. 43
2.2.8 Fluorescence Anisotropy........................................................................... 44
2.3 Results .............................................................................................................. 45
2.3.1 Peptide Preparation ................................................................................... 45
2.3.2 UV-visible spectrum of Fe(II)-NZF-1-F2F3............................................. 46
2.3.3 Addition of Co(II) to Fe(II)-NZF-1-F2F3 ................................................. 46
2.3.4 Air Oxidation of Fe(II)-NZF-1-F2F3 ........................................................ 47
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2.3.5 DNA Binding of M-NZF-1-F2F3 to -RARE DNA ................................ 48
2.4 Discussion ........................................................................................................ 51
2.5 References ........................................................................................................ 56
Chapter 3. Switching Metal Ion Coordination and DNA Recognition in a Tandem
CCHHC-type Zinc Finger Peptide ................................................................................ 61
3.1 Introduction ...................................................................................................... 61
3.2 Materials and Methods ..................................................................................... 65
3.2.1 Nomenclature ............................................................................................. 65
3.2.2 Expression and Purification of NZF-1-F2F3 and MyT1-F2F3 ................. 65
3.2.3 Design of NZF-1-F2F3 and MyT1-F2F3 Mutants .................................... 66
3.2.4 Metal Binding Studies ............................................................................... 67
3.2.5 Circular Dichroism .................................................................................... 67
3.2.6 Oligonucleotide Probes ............................................................................. 68
3.2.7 Fluorescence Anisotropy ........................................................................... 68
3.2.8 Generation of Sequence Logos .................................................................. 69
3.3 Results and Discussion..................................................................................... 70
3.3.1 Functional Roles of the Histidine Ligands within Each ZF of NZF-1 ...... 70
3.3.2 Metal Binding Studies ............................................................................... 72
3.3.3 Co(II) Direct Titrations ............................................................................. 73
3.3.4 Zn(II) Titrations ......................................................................................... 74
3.3.5 CD Spectra of NZF-1-F2F3, CCFHC and CCHFC Mutants .................... 75
3.3.6 DNA Binding Studies ................................................................................ 76
3.3.7 Switching the DNA Binding Properties of MyT1 to those of NZF-1 ....... 79
3.3.8 Metal Binding and Folding Studies ........................................................... 81
3.3.9 DNA Binding Studies of MyT1-F2F3 with β-RAR .................................. 82
3.4 Conclusions ...................................................................................................... 83
3.5 References ........................................................................................................ 86
Chapter 4. A role for hydrogen bonding in DNA recognition by the non-classical
CCHHC type zinc finger, NZF-1 ................................................................................... 96
4.1 Introduction ...................................................................................................... 96
4.2 Materials and Methods ..................................................................................... 99
4.2.1 Design of NZF-1-F2F3 Mutant Peptides................................................... 99
4.2.2 Expression and Purification of NZF-1-F2F3 Mutant Peptides ............... 100
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4.2.3 Metal Binding Studies ............................................................................. 101
4.2.4 Electron Paramagnetic Resonance (EPR) Spectroscopy ......................... 102
4.2.5 Oligonucleotide Probes ........................................................................... 103
4.2.6 Fluorescence Anisotropy (FA) ................................................................ 103
4.2.7 Generating PyMOL Models .................................................................... 104
4.3 Results and Discussion................................................................................... 105
4.4 References ...................................................................................................... 112
Chapter 5. A Designed Zinc Finger Peptide with Hydrolytic Activity .................... 117
5.1 Introduction .................................................................................................... 117
5.2 Materials and methods ................................................................................... 121
5.2.1 Identification of consensus sequence for CCHH ZFs (CP-1 update) ...... 121
5.2.2 Peptide preparation .................................................................................. 121
5.2.3 UV-visible metal binding titrations ......................................................... 122
5.2.4 Nuclear magnetic resonance (NMR) spectroscopy ................................. 123
5.2.5 Hydrolysis of 4-nitrophenyl acetate (4-NA) ........................................... 123
5.3 Results and discussion ................................................................................... 125
5.3.1 Revisiting the consensus peptide ............................................................. 125
5.3.2 Modifying CP-1 to promote hydrolytic activity ...................................... 126
5.3.3 CP-1(CAHH) and coordination of Co(II) ............................................... 128
5.3.4 Zn(II) coordination of CP-1(CAHH) ...................................................... 133
5.3.5 Secondary structure of CP-1(CAHH) upon metal ion coordination ....... 135
5.3.6 Hydrolysis of 4-nitrophenyl acetate (4-NA) ........................................... 137
5.4 Conclusions .................................................................................................... 141
5.5 References ...................................................................................................... 143
Chapter 6. Engineering a Zinc Finger Protein as an Artificial RNA Cleaving Agent:
Tristetraprolin-Ribonuclease 4 (TTP2D-RNase4) ..................................................... 155
6.1 Introduction .................................................................................................... 155
6.2 Materials and Methods ................................................................................... 160
6.2.1 Design of TTP2D-RNase4/RNase4 Constructs ...................................... 160
6.2.2 Expression and Purification of TTP2D-RNase4/RNase4 Constructs ..... 162
6.2.3 Metal Binding Analysis: UV-visible Spectroscopy ................................ 163
6.2.4 Metal Binding: Inductively Coupled Plasma Mass spectrometry ........... 164
6.2.5 Circular Dichroism(CD) .......................................................................... 164
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6.2.6 Analysis of TTP2D-RNase4 activity ....................................................... 165
6.3 Results and Discussion................................................................................... 166
6.3.1 Design and Construction of TTP2D-RNase4 ......................................... 166
6.3.2 Expression and Purification of TTP2D-RNase4/RNase4 ....................... 167
6.3.3 Co(II) Binding of TTP2D-RNase4 .......................................................... 168
6.3.4 Zn(II) Stoichiometry of TTP2D-RNase4 ................................................ 169
6.3.5 Folding of TTPRNase4 Constructs ......................................................... 170
6.3.6 RNA Hydrolysis by TTP2D-RNase4 ...................................................... 171
6.4 Conclusions .................................................................................................... 172
6.5 References ...................................................................................................... 174
Chapter 7. Conclusions and Future Directions .......................................................... 185
7.1 Zinc Finger Proteins ....................................................................................... 185
7.2 Neural Zinc Finger Factor/Myelin Transcription Factor Zinc Fingers .......... 186
7.2.1 Metal Binding of the NZF/MyT Family.................................................. 186
7.2.2 Role of the Non-Metal Coordinating Histidine Residues in NZF-1 ....... 188
7.2.3 Fold of the ZF domains of the NZF/MyT Family ................................... 189
7.2.4 DNA Binding by the NZF/MyT Family.................................................. 190
7.3 Catalytic ZF Proteins ..................................................................................... 191
7.3.1 Consensus Peptide 1 (CP-1) .................................................................... 191
7.3.2 TTP2D-RNase4 ....................................................................................... 192
7.4 Future Directions for the NZF/MyT Family .................................................. 192
7.5 Future Directions for Catalytic ZFs ............................................................... 195
7.5.1 CP-1 ......................................................................................................... 195
7.5.2 TTP2D-RNase4 ....................................................................................... 195
7.6 References ...................................................................................................... 196
Appendix I. Characterization of the C-terminal Zinc Finger Clusters of the Neural
Zinc Finger Factor family: metal ion coordination and DNA binding .................... 206
I.1 Introduction..................................................................................................... 206
I.2 Materials and Methods ................................................................................... 211
I.2.1 Expression and Purification of NZF-1-F456 and MyT1-F467 ................ 211
I.2.2 Metal Binding Studies .............................................................................. 213
I.2.3 Fluorescence Anisotropy (FA) Studies .................................................... 213
I.3 Results and Discussion ................................................................................... 215
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I.3.1 Expression and Purification of NZF-1-F456 and MyT1-4567 ................ 216
I.3.2 Metal Binding Analysis ........................................................................... 217
I.3.3 DNA Binding to the βRAR Promoter ...................................................... 219
I.4 Conclusion ...................................................................................................... 221
I.5 References ....................................................................................................... 223
Comprehensive List of References .............................................................................. 236
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List of Tables
Table 1.1 Diseases/Disorders associated with the NZF/MyT family of proteins. ............. 9
Table 4.1 Upper limit dissociation constants for Co(II), Zn(II), and DNA binding to
NZF-1F2F3 WT and mutant peptides along with their dissociation constants for binding
to βRAR .......................................................................................................................... 107
Table 5.1 Second order rate constants (k'') of the hydrolysis of 4-NA by various
complexes and peptides. ................................................................................................. 139
Table 6.1 ICP-MS Analysis of TTP2D-RNase4, RNase4, and varients. ....................... 169
Table I.1 Kds of NZF-1 and MyT1 constructs for Co(II) and Zn(II) ............................. 219
Table I.2 Kd of NZF/MyT constructs for βRAR. .......................................................... 220
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List of Figures
Figure 1.1 Cartoon depicting the role of Zn(II) in promoting the structure of ZFs. .......... 2
Figure 1.2 Alignment and sequences of ZF domains of the NZF/MyT Family ................ 3
Figure 1.3 Metal Binding Analysis of NZF/MyT1 Family. ............................................. 10
Figure 1.4 Effects of alternate histidine coordination on d-d transition envelope in the
optical spectrum.. .............................................................................................................. 11
Figure 1.5 Hydrogen bond analysis of NZF-1.. ............................................................... 13
Figure 1.6 Structural analysis of the NZF/MyT family.. ................................................. 14
Figure 1.7 DNA targets of the NZF/MyT family............................................................. 16
Figure 1.8 DNA binding studies of MyT1 ....................................................................... 17
Figure 1.9 Published model of MyT1 F4 + F5 interaction with βRAR DNA ................. 18
Figure 2.1 Clustering of CCHHC zinc binding domain................................................... 37
Figure 2.2 Absorption spectrum of Fe(II)-NZF-1-F2F3 .................................................. 39
Figure 2.3 UV visible spectrum of two equivalents of Co(II) added into 50 µM Fe(II)NZF-1-F2F3 ...................................................................................................................... 46
Figure 2.4 Sequences of the fluorescein labeled -RARE and nonspecific probes used for
fluorescence anisotropy studies ........................................................................................ 47
Figure 2.5 Change in anisotropy (as fraction bound) upon the addition of Zn(II)-NZF-1F2F3 to -RARE DNA ..................................................................................................... 48
Figure 2.6 Change in anisotropy (as fraction bound) upon the addition of Fe(II)-NZF-1F2F3 to -RARE DNA ..................................................................................................... 49
Figure 2.7 Change in anisotropy (as fraction bound) upon the addition of Fe(II)-NZF-1F2F3 to -RARE DNA ...………………………………………………………………..50
Figure 3.1 Alignment and Structures of NZF-1 and MyT1 ............................................. 63
Figure 3.2 Co(II) and Zn(II) titration of CCFHC............................................................. 71
Figure 3.3 Co(II) and Zn(II) titration of NZF-1-F2F3 ..................................................... 72
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Figure 3.4 Co(II) and Zn(II) titration of CCHFC............................................................. 73
Figure 3.5 Comparison of NZF-1-F2F3 and Mutant Metal Binding and Fold. ............... 74
Figure 3.6 Comparison of apo-, Co(II)-, and Zn(II)-Bound NZF-1-F2F3 Peptides ........ 75
Figure 3.7 Fluorescence Anisotropy of NZF-1-F2F3 Variants........................................ 76
Figure 3.8 Model of mMyT1 interacting with the βRAR DNA ...................................... 77
Figure 3.9 Co(II) and Zn(II) titration of MyT1-F2F3 ..................................................... 78
Figure 3.10 Co(II) and Zn(II) titration of Q291R MyT1-F2F3 ....................................... 79
Figure 3.11 Comparison of Fold of MyT1-F2F3 Variants and NZF-1-F2F3.. ................ 80
Figure 3.12 Comparison of apo-, Co(II)-, and Zn(II)-Bound MyT1-F2F3 Peptides ....... 81
Figure 3.13 Fluorescence Anisotropy of MyT1-F2F3 Variants ...................................... 82
Figure 3.14 Comparison of Amino Acids in CCHHC and CCHH Domains ................... 84
Figure 4.1 Zinc Finger Domains of the NZF Family. ...................................................... 97
Figure 4. 2 HPLC Purified Peptides............................................................................... 101
Figure 4.3 Co(II) and Zn(II) titration of H515Q. ........................................................... 105
Figure 4.4 Co(II) and Zn(II) titration of H559Q and H515/559Q ................................. 106
Figure 4.5 Metal Center and DNA Binding Analysis of NZF-1-F2F3 Variants ........... 107
Figure 4.6 EPR Analysis of Double Mutants ................................................................. 108
Figure 4.7 Hydrogen Bond Analysis.............................................................................. 109
Figure 5.1 Consensus peptide-1 (CP-1) constructs . ...................................................... 118
Figure 5.2 Comparison of UV-Visible Spectra of CCHH vs CAHH CP-1 ................... 129
Figure 5.3 Co(II) Titration of CP-1(CAHH) .................................................................. 132
Figure 5.4 Zn(II) Binding of CP-1(CAHH) ................................................................... 135
Figure 5.5 Hydrolysis of 4-NA. ..................................................................................... 136
xiv
Figure 5.6 Formation of 4-NP over Time.. .................................................................... 137
Figure 5.7 Second Order Rate Constants of the Reaction with 4-NA. .......................... 138
Figure 6.1 NMR Solution Structure of Tis11d, a TTP Homolog .................................. 156
Figure 6.2 ZF Domains of TTP ...................................................................................... 157
Figure 6.3 Representation of ZFN. ZFN ........................................................................ 158
Figure 6.4 Model of TTP Ribonuclease Strategy .......................................................... 159
Figure 6.5 Purification of TTP2D-RNase4. ................................................................... 167
Figure 6.6 Co(II) Binding of TTP2D-RNase4. .............................................................. 168
Figure 6.7 CD Spectra of TTP2D-RNase4 Variants ...................................................... 170
Figure 6.8 Representation of FRET Assay. ................................................................... 171
Figure 6.9 RNA Hydrolysis by TTP2D-RNase4 ........................................................... 172
Figure I.1 Sequence Logo of ZF Domains. ................................................................... 207
Figure I.2 Sequence of CCHC type ZF domains ........................................................... 208
Figure I.3 Sequence Logo of CCHHC ZF Domains.. .................................................... 209
Figure I.4 NMR Solution Structures of NZF-1 and MyT1 ............................................ 209
Figure I.5 Sequence Logo of DNA Target Sequences................................................... 210
Figure I.6 Expression of NZF-1-F456 and MyT1 F4567. ............................................. 215
Figure I.7 Purification of NZF-1-F456 and MyT1-4567 ............................................... 216
Figure I.8 Co(II) and Zn(II) Titration of NZF-1-F456. ................................................. 217
Figure I.9 Co(II) and Zn(II) Titration of MyT1-F4567 ................................................. 218
Figure I.10 FA of NZF-1-F456 and MyT1-F456 .......................................................... 220
Figure I.11 Percent identity of NZF-1 and MyT1 ......................................................... 221
xv
Abbreviations
4-NA
4-Nitrophenyl Acetate
4-NP
4-Nitrophenolate
AML
Acute Myloid Leukemia
ARE
AU-Rich Element
βRAR
β Retinoic Acid Receptor
βRARE
β Retinoic Acid Response Element
BSA
Bovine Serum Albumin
CD
Circular Dichroism
CNP
2’3’-cyclic-nucleotide 3’phosphodiesterase
CNS
Central Nervous System
CP-1
Consensus Peptide – 1
Cy3
Cyanine-3
DTT
Dithiothreitol
EDTA
Ethylenediaminetetraacetic Acid
EFRET
Fluorescence Resonance Energy Transfer Efficiency
EMSA
Electrophoretic Mobility Shift Assay
EPR
Electron Paramagnetic Resonance
ER
Estrogen Receptor
FA
Fluorescence Anisotropy
Fl
Fluorescein
FRET
Fluorescence Resonance Energy Transfer
FTase
Farnesyltransferase
xvi
GST
Glutathione-S-Transferase
HDAC
Histone Deacetylase
HDR
Homology Directed Repair
HPLC
High Performance Liquid Chromatography
ICP-MS
Inductively Coupled – Mass Spectrometry
IL
Interleukin
IPTG
Isopropyl β-D-1-Thiogalactopyranoside
ITC
Isothermal Titration Calorimetry
Kd
Dissociation Constant
LB
Luria Bertani
LFSE
Ligand Field Stabilization Energy
LMCT
Ligand to Metal Charge Transfer
LSD1
Lysine-Specific Demethylase 1
MBNL
Muscleblind-Like
MES
2-(N-morpholino)ethanesulfonic acid
MyT1
Myelin Transcription Factor 1
NHEJ
Non-Homologous End Joining
NMR
Nuclear Magnetic Resonance
NZF-1
Neural Zinc Finger Factor – 1
PCR
Polymerase Chain Reaction
PDF
Peptide Deformylase
PLP
Proteolipid Protein
RNAi
RNA Interference
xvii
RNase4
Ribonuclease 4
shRNA
Short Hairpin RNA
siRNA
Small Interfering RNA
SNP
Single Nucleotide Polymorphism
Sp1
Specificity Protein 1
SPR
Surface Plasmon Resonance
ST18
Suppression of Tumorigenicity 18
TCEP
Tris(2-carboxyethyl)phosphine
TFA
Trifluoroacetic Acid
TFIIIA
Transcription Factor III A
TNFα
Tumor Necrosis Factor α
TTP
Tristetraprolin
UTR
Untranslated Region
Vo
Initial Reaction Velocity
WT
Wild Type
ZF
Zinc Finger
ZFN
Zinc Finger Nuclease
xviii
Chapter 1
The Neural Zinc Finger Factor/Myelin Transcription factor proteins: a
unique family of “non-classical” zinc finger proteins*
1.1 Introduction
Metal ions, which are required for a myriad of biological processes, are found as
cofactors in more than a third of structurally characterized proteins (1-6). Zinc, in
particular, plays diverse roles in the body, such as serving as a cofactor in up to 10% of
all proteins and acting as a signaling molecule (7-11). Zinc finger (ZF) proteins represent
a large family of zinc co-factored proteins that comprise 3 – 5% of the human genome (7,
12-16). More than 14 different classes of ZF proteins have been identified, all of which
coordinate zinc ions in a tetrahedral geometry using a combination of four cysteine
and/or histidine residues (13-23). The classes of ZF proteins are delineated based on the
exact composition of the metal coordinating ligand set (e.g. the number and sequence of
cysteine and histidine residues), the spacing between these ligands, as well as the fold
that the protein adopts once metal is bound (14, 16). In the absence of metal ions, these
domains lack secondary structure, but adopt structure and thus function when zinc ions
are coordinated (Figure 1.1) (23). These unique and ubiquitous proteins have various
functions including transcriptional and translational control (7, 14, 16, 23). Of particular
interest is their ability to interact with DNA, as over half of all human transcription
* Adapted from the publication: Besold, A.N.; Lee, S.J.; Sue Lue, N.; Cymet, H.J. ;
Michel, S.L.J. 2010. J. Biol.Inorg. Chem, 15(4):583-90.
1
Figure 1.1 Cartoon depicting the role of Zn(II) in promoting the structure of ZFs. In
the absence of Zn(II), the ZF domain is unstructured. Upon zinc coordination, the domain
adopts distinct secondary strucutre. In the case of “classical” ZFs (CCHH), such as XFIN
(PDB: 1ZNF), this involves a ββα fold.
factors contain ZF domains (24, 25). The most well studied DNA binding ZFs are the
‘classical’ ZF proteins, which have Cys2His2 domains (CCHH motif) and adopt a ββα
fold upon zinc coordination (13, 14, 16). The DNA binding properties of these classical
ZFs are so well understood that they are the focus of many protein design efforts to
engineer artificial agents for gene therapy (24, 26-29). The ‘non-classical’ ZF proteins are
less well studied and the data obtained to date has revealed that these proteins adopt a
large range of secondary structures upon zinc coordination (15, 16). The focus of this
‘topics’ article is the Neural Zinc Finger/Myelin Transcription Factor (NZF/MyT) family
of non-classical ZF proteins, which are essential to the development of the central
nervous system (CNS). These proteins contain unique sequence and structural elements
that are critical for their function (16, 30-32).
The NZF/MyT family of ZF proteins is a small, but critically important family of
ZFs. Three members have been identified: Myelin Transcription Factor 1 (MyT1 or NZF2), Neural Zinc Finger Factor-1 (NZF-1 or MyTl-like or png-1) and Suppression of
Tumorigenicity 18 (ST18 or NZF-3) (30-32).
2
These proteins contain multiple ZF
domains that are found in clusters of 1, 2, 3, or 4 (Figure 1.2a). Each of these domains
contains five absolutely conserved cysteine and histidine residues with the sequence
CPXPGCXGXGHX7HRX4C (Figure 1.2b and 1.2c), where X is any amino acid
(CCHHC motif). There is little variability in the amino acid sequence in these zinc sites,
with domains between proteins showing upwards of 100% identity (Figure 1.2c) (16).
This is unusual as classical ZF domains typically have high conservation only in the
metal coordinating residues as well as a few additional amino acids which are important
for protein structure (CX2-5CX12-13HX3-5H) (33). MyT1 was the first CCHHC ZF to be
identified (31). Identification came from a study aimed at discovering proteins that
control the differentiation of glial cells, such as oligodendrocytes and astrocytes, from
progenitor cells. Specifically, a lambda phage cDNA expression library derived from
(a)
(b)
(c)
Figure 1.2 Alignment and sequences of ZF domains of the NZF/MyT Family. (a)
Cartoon of ZF clusters in each protein of Homo sapiens. (b) Alignment of ZF2 from
each H. sapiens protein. (c) Sequence logo depicting conservation of amino acids. The
height corresponds to degree of conservation, with 4 bits being 100% conserved.
3
human fetal brain was screened for transcription factors that bound to a cis-regulatory
element in the proteolipid protein (PLP) promoter. PLP is the main myelin forming
protein in the CNS and is one of the final targets of regulation in oligodendrocytes,
serving as a good target gene to use to identify essential transcription factors in glial cell
development (31, 34). This study identified MyT1 as the transcription factor responsible
for binding to the PLP promoter. Subsequently, NZF-1 was discovered using the same
approach, but with the goal of identifying proteins that bound to retinoic response
elements. Specifically, these studies aimed to identify proteins that bound to the promoter
of β-retinoic acid receptor (βRAR), a transcription factor responsible for regulating genes
of various functions such as those essential for the control of cell growth and
differentiation, and the Pituitary-Specific Positive Transcription Factor 1 (Pit-1), which is
essential for the development of the pituitary gland and for hormone production (30, 3537). Given the emerging importance of this family of proteins, two years after the
discovery of NZF-1, an attempt to find additional members of this family was made.
Using primers corresponding to conserved regions of NZF-1 and MyT1, similar DNA
sequences from various human tissues was isolated using the polymerase chain reaction.
From these studies, ST18 was cloned and isolated and found to be enriched in the brain
(32).
1.2 Biological Role
All members of the NZF/MyT family of proteins are found primarily in the CNS
where they control the differentiation of neuronal cells including neurons and
oligodendrocytes (30-32, 38). NZF-1 is found predominantly in neurons and is one of a
4
handful of proteins shown to be necessary for the formation of neurons from fibroblast
and adipose progenitor cells (39-44). This role was later clarified when NZF-1 was
shown to enhance the maturation of neuronal cells (45). NZF-1 promotes neuronal
development by activating the expression of βRAR, which regulates several biological
processes including cell growth and differentiation (30, 35, 37). NZF-1 is also present in
the pituitary gland, where it can bind to the Pit-1 gene, which encodes the Pit-1 protein
which is important for activating growth hormone and prolactin genes (30, 36). In the
CNS, NZF-1 expression is highest during development and decreases in adulthood (30,
46). The opposite is true in the pituitary gland, where expression is highest in adults. Of
note, an alternatively spliced form of NZF-1 is found in the testes, but the role of NZF-1
in this organ has not been explored (30). Thus, NZF-1 plays essential roles throughout
life by regulating different genes important for the development and function of the CNS
and pituitary gland.
Unlike NZF-1, MyT1 is found predominantly in oligodendrocytes, where it activates
PLP expression, which forms myelin (31). In Xenopus leavis, MyT1 has been shown to
be essential in both retinal and neural differentiation (47). In the retina, low levels of
MyT1 are needed to activate the protein Xath5, which is essential for the development of
neurons located near the inner surface of the retina. After initial activation of Xath5, this
protein further activates the expression of MyT1 leading to retinal growth. In neurons,
MyT1 promotes growth by acting as a repressor. Specifically, MyT1 is part of a complex
with Lysine-Specific Demethylase 1 (LSD1), which demethylates lysines of histones to
control transcription, CoREST, which acts as co-repressor with LSD1, and Histone
Deacetylase 1/2 (HDAC1/2), which controls transcription by deacetylating lysines of
5
histones. This complex negatively regulates the expression of Pten, which is a protein
responsible for decreasing cell proliferation. This negative regulation counteracts the
effect of Pten and enhances neuronal growth (48). MyT1 has also been identified as
important in the development of the peripheral nervous system of the invertebrate
chordate, Ciona intestinalis (49). MyT1 is also highly expressed in the pancreas where it
is involved in islet cell formation (50-52). Interestingly, if MyT1 is deleted in this organ,
there is an increase in the expression of the other homologues of this family, suggesting
possible cross-talk in their functions (51). Thus, despite the dual roles of MyT1 as an
activator and a repressor of transcription, this CCHHC protein always works to promote
cell growth and differentiation.
The third member of this family of proteins, ST18, is not well understood in terms of
it biological function. It is highly expressed in the brain, but is also present in several
other tissues including the heart, kidney, liver, eye, testis, ovaries, prostate, thyroid, aorta,
and stomach (53). Downregulation of ST18 expression results in a change in the mRNA
levels of pro- and anti- inflammatory as well as pro- and anti- apoptosis genes in
fibroblast cells, suggesting ST18 plays a role in these pathways (54). The majority of
these genes are pro-inflammatory and pro-apoptotic and these effects are potentially a
result of ST18’s regulation of tumor necrosis factor α (TNFα) (54). Studies have shown
that nuclear extracts that contain ST18 are able to bind to the promoter region of TNFα
and ST18 upregulates TNFα mRNA in fibroblast cells. ST18 is also expressed in the
pancreas, where it mediates lipotoxicity and apoptosis of β cells (55). Thus, ST18 appears
to play a variety of roles in the body, but many of its biological functions have not yet
been defined.
6
All members of the CCHHC ZF family are present in the nucleus of cells when active
(32, 46, 56). Nuclear expression of NZF-1 and MyT1 is highest during development, with
low levels of the proteins typically found in adults, with the exception of NZF-1, which is
found in high levels in the adult pancreas (30, 57). As PLP accumulates in
oligodendrocytes, MyT1 is trafficked to the cytoplasm, presumably as means to quench
its activity, where levels of this protein eventually become undetectable (57). When
present in the cytoplasm, NZF-1 and MyT1 can interact with the plasma membrane
protein Lingo-1, which likely holds these proteins in the cytoplasm in order to render
them inactive (58). Recently, Lingo-1 expression has been associated with decreased
myelination, potentially by detrimentally regulating MyT1 activity (59). Less is known
about the localization of ST18 in the nervous system, but this protein is present in the
cytoplasm of the pancreas and only shifts to the nucleus when it is induced by factors
such as fatty acids (55).
1.3 Regulation of CCHHC ZF Proteins
In addition to the regulation of NZF-1 and MyT1 by Lingo-1, other factors have also
been shown to control the expression and activity of the NZF/MyT family. Regulation of
MyT1 in the CNS is mostly widely studied in X. leavis development. In this organism,
pro-neural genes NGN-1 and NGNR-1 activate the transcription of MyT1 (60, 61). Low
levels of MyT1 are required for Xath5 expression, which then further enhances MyT1
expression (47). In mammals, the growth factor bFGF enhances MyT1 expression while
Sin3B, which negatively regulates transcription through the recruitment of HDACs,
decreases MyT1 activity and potentially the activity of NZF-1 as well (62, 63).
7
Expression of NZF-1 is upgregulated in retinoic acid induced cells, which is interesting
given that NZF-1 regulates the expression of βRAR, which requires retinoic acid to
function (64).
Less is known about how these proteins are regulated in other organs, but in the
pancreas NKX6, a transcription factor required for β-cell development, regulates MyT1
expression (65). Another pancreatic protein, manic fringe, increases the expression of
both MyT1 and ST18 (66). More research needs to be done to fully understand how these
proteins are regulated, which is essential given that misregulation of these proteins has
been linked to a number of disorders.
1.4 Disease States Linked to NZF/MyT Proteins
Table 1.1 summarizes the diseases/disorders that occur when NZF/MyT proteins are
either misregulated or deleted (53, 67-90). Typically, misregulation/deletion is
detrimental. For example, deletion of the MyT1 or NZF-1 genes is linked to intellectual
disability, although how the brain develops in these patients as a result of these deletions
is unclear (72, 73, 81). Upregulation of NZF-1 and ST18 have been linked to
oligodendroglioma and Acute Myloid Leukemia (AML), respectively. The link to
oligodendroglioma is particularly interesting as this type of brain cancer originates from
oligodendrocytes, where NZF-1 is not normally expressed. There are a few documented
cases in which misregulation of these proteins are beneficial. For example, a single
nucleotide polymorphism (SNP) of NZF-1 is associated with a better outcome in gastric
cancer (75). Upregulation of MyT1 has been observed in areas of multiple sclerosis
lesions associated with remyelination (80). Interestingly, upregulation of MyT1
8
Table 1.1 Diseases/Disorders associated with the NZF/MyT family of proteins.
Protein
NZF-1
Disease/Disorder
Oligodendroglioma
ADHD
Schizophrenia
Depression
Intellectual Disability
MyT1
ST18
Multiple Sclerosis
Gastric Cancer (beneficial)
Spinal Injury (beneficial)
Perventricular Leukomalacia
Schizophrenia
Multiple Sclerosis (beneficial)
Intellectual Disability
Cocaine Addication (beneficial)
Alzheimer's
Alcoholism
Breast Cancer
Acute Myloid Leukemia
Down Syndrome-Megakaryoblastic Leukemia
Alcoholism
Glaucoma
Assocation
Upregulation
SNP rs2241685
Duplication
Alternate Gene Regulation
SNP rs3748989
SNP rs1617213
SNP rs6759709
Gene Deletion
Deletion or Duplication
SNP rs2053906
SNP rs17039396
Upregulation
Upregulation
Alternate Gene Regulation
Upregulation
Gene Deletion
Upregulation
Alternate Gene Regulation
Upregulation
Gene Deletion
Upregulation
Downregulation
Downregulation
SNP rs1015213
Ref
(67)
(66)
(67)
(68)
(71)
(71)
(71)
(72)
(73)
(74)
(74)
(76)
(77)
(78-79)
(80)
(81)
(82-82)
(84)
(85)
(53)
(86)
(87)
(85)
(88)
expression has also been associated with decreased cocaine self-administration,
suggesting that induction of MyT1 may be a beneficial means to treat addiction (82, 83).
MyT1 is also upregulated in the brain of alcoholics, while ST18 is downregulated, but the
consequences of these changes in transcript levels are not known (85). ST18
misregulation has various consequences as this protein can act as an oncogene as well as
tumor suppressor (86, 87). ST18 is most often downregulated in disease states, which can
be a detriment given the tumor suppression activity of ST18, as is the case with Down
Syndrome – Megakaryoblastic Leukemia and breast cancer (53, 87). However, ST18 can
also be detrimental when this proteins acts as an oncogene, as is the case of AML (86).
The roles played by these proteins in these myriad of diseases has not yet been fully
delineated; however, proper regulation of these proteins is clearly important.
9
(a)
(b)
(c)
(d)
Figure 1.3 (a) Schematic representation of experimental procedure for UV-visible
monitored metal titrations. The apo-ZF is titrated with Co(II) and once saturated, the
Co(II)-ZF is titrated with Zn(II). (b) Example of d-d transition bands that form when Co(II)
binds to the CCHHC ZF domains during a titration, in this case F2 + F3 of MyT1. (c) An
example of the titration data that has been fit to a 1:1 binding model. Pink: Co(II) fit; Blue:
Zn(II) Fit. (d) NMR solution structure of F2 of NZF-1 (PDB: 1PXE)
1.5 Metal Ion Site: Coordinating Ligands
Metal ions are the required functional co-factors for the NZF/MyT family of proteins.
Five potential metal coordinating ligands are present in the ZF domains of this family,
arranged in a CCHHC motif (Figure 1.2b). This motif is unusual as ZFs typically require
only four coordinating ligands. To determine which of the five potential Zn(II) ligands
are involved in metal ion coordination, two approaches have been taken: UV-visible
spectroscopy and nuclear magnetic resonance (NMR). Zn(II) binding cannot be directly
assessed using UV-visible spectroscopy as Zn(II) is spectroscopically silent due to its d10
electron count. Thus, Co(II) is often used as a spectroscopic probe because Co(II)
coordinates ZF proteins in a similar manner as Zn(II), but has a d7 electron count
resulting in rich spectroscopic properties (91-93). Typically, when Co(II) is coordinated
to ZF sites, d-d transitions between 550 – 750 nm, indicative of tetrahedral geometry, are
observed (94). These d-d transitions are very sensitive to the environment at the Co(II)
10
Figure 1.4 Effects of alternate histidine coordination on d-d transition envelope in
the optical spectrum. Purple: Wild type CCHHC NZF-1, Blue: CCFHC NZF-1, Red:
CCHFC NZF-1.
center and can provide information regarding coordination number and ligand set (95).
Zn(II) binds more tightly to these sites than Co(II) because there is no ligand field
stabilization energy penalty for Zn(II) in tetrahedral versus octahedral geometries (Figure
1.3a – c) (17). Thus, Zn(II) binding can be monitored by following the disappearance of
d-d bands as Zn(II) is titrated with Co(II)-ZF [Zn(II) displaces the Co(II)] (16, 17). This
approach allows one to determine upper limit dissociation constants (Kds) for both Co(II)
and Zn(II) and it has been utilized for several of ZF domains within the NZF/MyT family
of proteins (21, 96-98). The d-d transitions that appear upon Co(II) binding for the
CCHHC ZF domains display absorbance maxima at 593, 646, and 679 nm, indicative of
tetrahedral geometry with a CCHC ligand set. Mutational analyses to determine which of
the conserved histidine residues coordinates metal have been reported by our group and
others (98, 99). When the second histidine residue is mutated to a glutamine or a
phenylalanne, the d-d transitions indicate retention of tetrahedral geometry with a CCHC
ligand set (Figure 1.4), but the shape of the d-d transitions differ, indicating the
environment at the metal center has been altered (98, 99). This is not observed when the
11
first histidine is mutated to a glutamine or phenylalanine, thus the mutagenesis/UVvisible spectroscopy studies revealed that the second histidine coordinates metal ions.
These results have been validated via the three NMR solution structures: F2 of NZF-1, F5
of MyT1, and F4+F5 of ST18 (96, 98-101). In all three structures, the second histidine is
shown to coordinate Zn(II) along with the three cysteine residues.
1.6 Affinities of Co(II) and Zn(II) for the CCHHC Domains of NZF-1 and MyT1
The affinity of the CCHHC ZF domains for metal ions has been assessed using both
UV-visible spectroscopy, as described above, and isothermal titration calorimetry (ITC)
(21, 96, 98). ITC, unlike UV-visible spectroscopy, does not require the use of Co(II) as a
spectroscopic probe. Using this method, Wilcox and co-workers determined a
dissociation constant in the low nanomolar regime for finger 2 (F2) of MyT1 and this
dissociation constant was validated via the Co(II) displacement method (21). This is in
contrast to the mid-picomolar regime that was determined using the Co(II) displacement
method for a construct comprised of F2+F3 of MyT1 (98). The tighter binding seen for
the two finger construct of MyT1 suggests that there may be coopertivity in metal
binding for these multiple ZF domains. Cooperative metal binding is possible in ZF
domains as this has been observed in peptide models of the treble clef CCCC-type ZFs
(102). In addition to dissociation constants, ITC also revealed that zinc binding is more
entropically favored and less enthalpically favored, which contrasts the classical ZFs for
which an equal contribution of entropy and enthalpy has been reported (21).
12
1.7 Role of Non-Coordinating Histidine in CCHHC Domains
The ubiquity of the non-coordinating histidine in each ZF domain of the NZF/MyT
family suggests that it plays an important structural and/or functional role. From the
NMR structure of F2 of NZF-1, it was predicted that the non-metal coordinating histidine
participates in a stacking interaction with a tyrosine residue helping to stabilize one of the
loops present in the folded protein, which then allows the protein to function (Figure
1.3d) (99). However, we reported that when this conserved non-metal coordinating
histidine is mutated to phenylalanine to retain stacking in a two domain construct of
NZF-1 (F2+F3), DNA recognition is abolished, indicating that the role of this histidine is
not simply to be involved in pi stacking, as equivalent DNA binding should have been
observed in the mutant case (98). Further evidence for a larger role for this non-metal
coordinating histidine came from sequence analysis. The tyrosine residue thought to be
important for pi stacking is not conserved in all CCHHC ZF domains. For instance, in F3
of NZF-1, this residue is an arginine. The presence of an arginine suggests hydrogen
bonding may be a key interaction for this amino acid position instead of pi stacking
(Figure 1.5) (103). This hypothesis was borne out in studies by our laboratory in which
(a)
(b)
Figure 1.5 Hydrogen bond analysis of NZF-1. (a) d-d transition envelope of WT
CCHHC NZF-1 (purple), CCFHC NZF-1 (blue) and CCQHC (orange). (b) Analysis of
possible hydrogen bonds, represented by a dotted line, to or from the non-metal
coordinating histidine residues in F2 and F3.
13
the non-coordinating histidine was mutated to a glutamine residue (104). Mutation of
histidine to glutamine in a single ZF domain of the two domain construct of NZF-1
(F2+F3) results in a peptide that still binds DNA with sequence specificity, albeit with a
weaker Kd. This decrease in affinity cannot be attributed to single finger binding by the
intact ZF domain as a single ZF construct of NZF-1 cannot interact with DNA and
instead suggest that hydrogen bonding is important for the structure and function of NZF1. Current work in the Michel laboratory is focused on defining additional factors that are
important for DNA recognition (104).
1.8 Fold of the CCHHC ZF Domains
The available structural data for the NZF/MyT family of proteins (F2 of NZF-1, F5 of
MyT1, and F4+F5 of ST18) reveals that the folded protein is composed of a series of
loops centered around the zinc ion (Figure 1.2d, 1.6a – b) (99, 100, 105). This contrasts
classical ZF proteins, in which the zinc bound form contains significant beta sheet and
alpha helix content (Figure 1.1) (23) The lack of alpha helix and beta sheet content
observed in the NMR structures of the CCHHC family has been confirmed in circular
dichroism (CD) studies of F2+F3 of NZF-1, F2 of MyT1, and F2+F3 of MyT1 (21, 98).
(a)
(b)
(c)
Figure 1.6 Structural analysis of the NZF/MyT family. (a) NMR solution structure of
F5 of MyT1 (PDB: 2JYD) (b) NMR solution structure of F4+F5 of ST18 (PDB: 2CS8).
(c) CD spectra of apo F2+F3 of NZF-1 (purple), Co(II) bound F2+F3 of NZF-1 (pink),
and Zn(II) bound F2+F3 of NZF-1 (blue).
14
In the apo form, the CD is representative of random coil, as evidenced by the large
negative signal at 195 nm. Upon addition of Co(II) or Zn(II), this signal becomes less
negative and an additional feature appears around 225 nm, indicating the protein has
folded, but remains largely coiled (Figure 1.6c). These domains only bind DNA when
they are bound to metal, thus the predominantly “loop” type structure that is adopted
upon metal coordination is clearly important for function (98, 106).
1.9 DNA Partners
Although there is an unusually high degree of sequence similarity among the
CCHHC ZF family members, each member recognizes and regulates different genes (30,
31, 54). MyT1 was discovered based upon its ability to recognize a specific sequence of
the
PLP
promoter,
termed
“site
4,”
which
has
the
following
sequence:
AAGGATCAGTTGGAAGTTTCCAGGACATCT TC (31). Likewise, NZF-1 was
identified based upon its ability to bind a sequence in the βRAR promoter:
AATTGGGTTCACCGAAAGTTCAC (30). The DNA sequences used to identify these
proteins each contain a common “AAGTT” sequence (bolded above). Given the high
degree of sequence similarity in these ZF domains and the repeated appearance of this
AAGTT sequence, it was proposed that all the clusters within the NZF/MyT proteins
recognize this DNA target (30, 31, 54, 61, 96). Examination of the DNA sequences for
which binding data has been published supports this: the AAGTT motif is largely
conserved, albeit with some variation (Figure 1.7) (30, 31, 54, 107-109). Interestingly,
the AAGTT sequence is not present in every DNA to which this family binds. For
instance, MyT1 binds specifically to the “Opalin-MUT” sequence, which has an
15
(a)
(b)
(c)
Figure 1.7 DNA targets of the NZF/MyT family. (a) Alignment of DNA targets
identified in the literature. The conserved AAGTT region is boxed. (b) Sequence logo of
DNA targets for which there is published binding data. Height corresponds to degree of
conservation with 2 bits being 100% conserved. (c) Sequence logo of DNA targets for all
identified DNA targets, including putative targets. Height corresponds to degree of
conservation with 2 bits being 100% conserved.
“AACCA” sequence in place of the “AAGTT,” suggesting other factors outside of these
five bases are important for sequence specific DNA interactions (108).
Exactly how this family of proteins recognizes DNA remains unresolved. To
better understand DNA recognition by the CCHHC family, we have recently examined
the DNA binding specificity of F2+F3 of NZF-1 and F2+F3 of MyT1. Using the βRAR
sequence in fluorescence anisotropy studies, we have demonstrated that F2+F3 of NZF-1
binds to this target sequence specifically with a Kd in the low nanomolar regime, while
F2+F3 of MyT1 binds to this sequence non-specifically (98, 106). The non-specific DNA
binding of F2+F3 of MyT1 was striking as this construct is 92% identical to F2+F3 of
16
(a)
(b)
Figure 1.8 DNA binding studies of MyT1. (a) Fluorescence anisotropy of wild type F2+F3
of MyT1 interacting with βRAR (red) and a random DNA segment (blue). (b) Fluorescence
anisotropy data of Q291R F2+F3 of MyT1 interacting with βRAR (red) and a random DNA
segment (blue).
NZF-1. Mutation of just one amino acid in this MyT1 construct, a glutamine, to the
amino acid present in that position of NZF-1, an arginine, resulted in MyT1 binding to
the βRAR sequence specifically with low nanomolar binding affinity (Figure 1.8) (98).
The amino acid in this important position is not conserved in this family of proteins
(Figure 1.1c position 9 in figure 1.1), suggesting a model of DNA recognition in which
the few non-conserved amino acids in the protein sequence drive sequence specific DNA
recognition.
To gain more insight into the DNA binding properties of this family, MacKay and
co-workers reported a HADDOCK modeling approach coupled with NMR which
allowed them to propose a mechanism for MyT1 binding to βRAR (Figure 1.9) (101).
While the βRAR gene is not the physiological target of MyT1, it does contain the AAGTT
sequence thought to be important for DNA recognition and thus these studies may
provide insight into how this family recognizes this important motif. The main feature of
the Mackay model is that the ZF domains of MyT1 fit into the major groove of DNA,
making contact with the AAGTT sequence. This binding is driven by the conserved
amino acids in the ZF domain, which are likely important for the conserved fold the
17
Figure 1.9 Published model of MyT1 F4 + F5 interaction with βRAR DNA (PDBID
2MF8).
protein (33). This model does not take into account the sequence selectively we have
observed as a result of the non-conserved amino acids with our DNA binding studies of
NZF-1 and MyT1. Thus, additional structural studies are needed to delineate the mode of
protein/DNA recognition to test the model proposed by Mackay and Co-workers.
1.10
Conclusion
The NZF/MyT family of proteins contains several features that are unique to this
class of ZFs. These include the presence of five potential metal binding ligands in each
ZF domain and a high degree of sequence similarity within these domains. Moreover, the
DNA binding specificity of the domains appears to be modulated by single amino acids.
The full mechanisms of zinc mediated DNA recognition remains unresolved and it is
likely that additional unique features will be identified. A clearer understanding of the
mechanism of action of these proteins will not only contribute to our fundamental
18
knowledge of metal mediated transcriptional regulation, but also has the potential to help
us better understand the biological role of these proteins in neuronal development.
1.11
Thesis
The research described in this thesis focuses on several ZF proteins and includes
biochemical studies to assess protein function as well as protein design to engineer new
functions. The bulk of my research involves the NZF/MyT family of proteins described
above and in Chapters 2 – 4. In addition, I have also worked on redesigning ZF domains
to produce catalytic centers. In one effort, in collaboration with Dr. David Goldberg’s
laboratory (JHU Chemistry), a classical ZF construct was altered to endow the protein
with hydrolytic activity, as described in Chapter 5. In a second effort, the non-classical
ZF protein tristetraprolin (TTP) was engineered to include a ribonuclease domain to
selectively cleave RNA targets. This work, described in Chapter 6, was done in
collaboration with Dr. Gerald Wilson’s laboratory at UMB School of Medicine
Chapter 2 of my thesis describes studies of iron(II) coordination to the ZF domains of
NZF-1. We show that this iron(II) coordination does not alter the ability of the protein to
interact with DNA, indicating flexibility with regards to metallation. Chapter 3 focuses
on the DNA binding properties of two domain constructs (ZF2 + ZF3) of NZF-1 and
MyT1. We discovered that a single non-conserved amino acid present in ZF3 of NZF-1 is
critical for DNA binding. We also discovered a role for a highly conserved, non-metal
coordinating histidine residue in hydrogen bonding (Chapter 4). Chapter 5 details the
work done in collaboration Dr. Goldberg’s lab at JHU. We demonstrate that mutagenesis
of a single metal coordinating ligand in a classical ZF domain produces a zinc site that
19
can hydrolyze a substrate. Chapter 6 details our collaborative project with the Wilson
laboratory aimed at creating ZF-ribonucleases that utilize TTP as the ZF targeting
domain. Chapter 7 summarizes my work. Finally, an appendix is attached that details the
isolation and biochemical characterization of the multi-ZF domains of NZF-1 and MyT1.
This data suggests that each ZF cluster in this family interacts differently with the
proposed target AAGTT DNA sequence.
20
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36
Chapter 2
Functional Characterization of Iron-Substituted Neural Zinc Finger Factor 1:
Metal and DNA Binding Affinities*
2.1 Introduction
Neural zinc finger factor -1 (NZF-1) is a eukaryotic transcription factor belonging
to a class of zinc finger proteins characterized by a unique Cys-X4-Cys-X4-His-X7-HisX5-Cys (CCHHC) sequence motif (2, 3). NZF-1 and its homologs MyT1 and NZF3/ST18 (2-6) each contain multiple copies of this CCHHC zinc binding domain that are
predominantly grouped into clusters of 2, 3 or 4 (Figure 2.1) (2-4, 6, 7). Collectively
referred to as NZF-1/MyT1 type zinc finger proteins, these transcription factors are found
principally in the nervous system and in parts of the endocrine system often during
development (4, 8-10).
NZF-1, in particular, plays a critical role in neuronal cell
NZF-1
MyT1
NZF-3/
ST18
CeMyT1
Figure 2.1 Clustering of CCHHC zinc binding domain. Zinc domains of NZF-1 and
some representative homologs show in black rectangles. NZF-1-F2F3 is bracketed.
* Adapted from the publication: Besold, A.N.; Lee, S.J.; Sue Lue, N.; Cymet, H.J. ;
Michel, S.L.J. 2010. J. Biol.Inorg. Chem, 15(4):583-90.
37
differentiation (2).
The CCHHC zinc binding domains mediate the DNA binding properties of this
protein family. NZF-1 recognizes a promoter element in the -retinoic acid receptor gene,
termed RARE (-retinoic acid receptor element) as well as two additional elements in
the mouse Pit-1 gene promoter (2). All known target DNA sequences for the NZF1/MyT1 proteins contain a central AGTT core. Both the 2-domain and 3-domain clusters
within NZF-1 are individually capable of specific binding to the RARE site (2, 11).
One hypothesis for NZF-1’s DNA binding promiscuity is that the high degree of
homology between the individual zinc fingers in NZF-1 allows the different domain
clusters to interact with the same DNA sequence. The ability to vary which CCHHC
domains bind the target gene promoter may be one mechanism by which these
transcription factors regulate gene transcription.
Zinc finger domains typically utilize a combination of four cysteine and/or
histidine ligands to coordinate zinc (12-16). Because NZF-1 has five potential zinc
binding ligands in its conserved sequence, Cys-X4-Cys-X4-His-X7-His-X5-Cys, one of the
first biophysical questions to be addressed regarding NZF-1 was the determination of the
ligand configuration around the zinc ion. The NMR structure of a single NZF-1 zinc
finger identified all three cysteine residues and the second conserved histidine as the zinc
ligands (17). The first conserved histidine appeared to stabilize an internal loop within
this structure through an aromatic interaction with a semi-conserved tyrosine (Figure
2.2). This same interaction was not observed in two subsequent NMR structures of NZF1 homologs (18, 19) and the role of this additional conserved histidine is still unknown.
The overall fold of the NZF-1 zinc finger lacks major secondary structural elements (17),
38
in contrast to other types of zinc finger proteins, such as classical zinc fingers which
contain an anti-parallel beta sheet followed by an alpha helix (12). While no clear DNAcontacting surface was revealed by the NZF-1 domain structure, a recently reported
model of a single MyT1 domain bound to DNA suggests a unique recognition mode in
which the entire zinc finger domain inserts itself into the major groove (19). Further
work is needed to confirm this model and importantly to determine the mechanism by
which multiple domains interact with DNA, as required for high affinity, specific DNA
binding.
Zinc finger proteins are presumed to favor zinc in vivo because there is no energy
penalty to be paid by ligand field stabilization energy (LFSE) for tetrahedral zinc (II) as
there is for other open shell metal ions (20). Despite this energetic preference for zinc, the
presence of mixed nitrogen and sulfur donor ligands allows zinc finger proteins to bind to
other metal ions, including iron. There are several examples of iron substituting for zinc
Figure 2.2 Solution structure of a single NZF-1 zinc binding domain (PDBID 1PXE). The
zinc coordinating ligands, 3 cysteines and 1 histidine, are shown, along with an apparent
stabilizing interaction between the additional conserved histidine and a semiconserved tyrosine.
Note the lack of secondary structure within the domain. Figure made in PyMol (17)
39
within the zinc-binding domains of zinc finger proteins, with varying effects on their
function (21-24). When iron is substituted for zinc in the classical zinc finger protein,
TFIIIA, the protein can no longer recognize its target DNA (23). The iron bound form of
the estrogen receptor (ER) zinc finger protein still binds to its target DNA but the
resultant complex is unstable (21). The iron-substituted GATA-1 zinc finger protein has a
higher affinity for DNA than its zinc bound counterpart (24). When iron binds to
Tristetraprolin, which is an RNA binding zinc finger protein involved in regulating
inflammatory response, similar RNA binding affinities are measured compared to the
zinc bound form of Tristetraprolin (22). Taken together, these findings suggest that iron
can have either a positive or negative effect on zinc finger protein function. The
functional effect of iron binding to NZF-1 has not been studied.
Iron has important roles within various cells of the nervous system. Iron
accumulation is associated with oligodendrocyte development (25), where it is needed for
cholesterol and lipid biosynthesis, key components of myelin. Oligodendrocytes also
have the highest rate of oxidative metabolism of any brain cell, contributing to their iron
requirement, presumably to maintain the myelin sheath (25). A high rate of iron uptake
and utilization by neurons has also been observed (26). Iron deposits have been localized
to regions of neural degeneration in Parkinson’s, Huntington’s, Alzheimer’s and ALS,
suggesting iron misregulation and iron-mediated oxidative stress during these disease
processes (26).
Given the significant roles played by NZF-1 and MyT1 in the developing nervous
system, the effect of iron coordination on their function is of interest. As such, we have
examined the iron binding properties of a construct of NZF-1, NZF-1-F2F3. NZF-1-F2F3
40
contains a cluster of two zinc binding domains and is the minimal fragment capable of
binding to DNA with high affinity. Using UV-visible spectroscopy we have determined
that iron can bind to the peptide in the ferrous state. We have used fluorescence
anisotropy to compare the abilities of M-NZF-1-F2F3, where M = Zn(II), Fe(II) or apo,
to selectively bind to the target -RARE DNA sequence. We discovered that the ferrous
bound peptide exhibited similar DNA binding affinity and selectivity as the zinc bound
form; while apo NZF-1-F2F3 was incapable of binding DNA.
2.2 Materials and methods
2.2.1 Preparation of NZF-1-F2F3
A fragment of rat NZF-1 corresponding to residues 487-584 (NZF-1-F2F3) was cloned,
expressed and purified as previously described (11). The NZF-1-F2F3 peptide has the
amino acid sequence MHVKKPYYDPSRTEKRESKCPTPGCDGT GHVTGLYPHHR
SLSGCPHKDRVPPEILAMHENVLKCPTPGCTGRGHVNSNRNSHRSLSGCPIAAAE
KLAKA, containing zinc binding domains 2 and 3 of the full-length protein (underlined).
Upon purification, NZF-1-F2F3 was maintained in an anaerobic environment for all
studies (95% N2, 5% H2).
2.2.2 Metal-Binding Titrations
Metal binding titrations were performed in either Teflon-top or screw-cap quartz cuvettes
using a Perkin-Elmer Lambda 25 UV/visible spectrometer. Titrations were performed in
200 mM HEPES, 100 mM NaCl at pH 7.4, prepared using metal-free reagents and water
that had been purified using a MilliQ purification system and passed over Sigma Chelex
41
resin.
Upon preparation, buffers were purged with helium or argon to degas and
transferred into a Coy inert atmospheric chamber. The following metal salts were used:
cobalt(II) chloride (EM Science); zinc atomic absorption standard (Aldrich; 15.2 mM
Zn2+ in 0.9% HCl) and (NH4)2Fe(SO4)2·6H2O (Aldrich). All stock solutions of metal
salts were prepared and maintained anaerobically. Peptide concentrations used in titration
experiments were typically 50 M and titrations were carried out in triplicate.
2.2.3 Co(II) and Zn(II) binding
The affinity of NZF-1-F2F3 for Co(II) was determined spectrophotometrically (11).
Titrations were performed in 200 mM Hepes, 100 mM NaCl at pH 7.4. Co(II) was
titrated into a solution of apopeptide until saturation of the d-d transitions. The relative
affinity of the peptide for Zn(II) was determined by monitoring the displacement of
Co(II) by Zn(II) (20). A dissociation constant for cobalt binding was determined by
fitting the data to a 1:1 binding model and using non-linear least squares analysis
(KaleidaGraph software, Synergy Software).
2.2.4 Fe(II) binding
A complete titration of NZF-1-F2F3 with Fe(II) was not performed owing to the
appearance in the titration spectra of additional bands between 240 and 400 nm upon the
addition of approximately 10 equiv of (NH4)2Fe(SO4)2. The UV–vis spectrum of the
Fe(II)–peptide complex in 200 mM HEPES, 100 mM NaCl at pH 7.4 was obtained by the
addition of 2 equiv of metal, sufficient to fill the metalbinding sites, in the presence of
150 mM sodium dithionite to ensure that the Fe(II) was completely reduced.
42
2.2.5 Displacement of Fe(II) by Co(II)
NZF-1-F2F3 (50 M) was reconstituted with 2 equivalents of Fe(II) in 200 mM Hepes
(pH 7.4)/100 mM NaCl/150M sodium dithionite. Cobalt(II) was added incrementally to
M-NZF-1-F2F3 [M= Fe(II) or Fe(III)] and the UV-visible spectrum was recorded after
each addition. Spectra with d-d transitions indicative of Co(II)-NZF-1-F2F3 were
observed.
2.2.6 Air oxidation of Fe(II)-NZF-1-F2F3
A solution of Fe(II)-NZF-1-F2F3 ( 50 M, 200 mM Hepes, 100 mM NaCl, 150 M
sodium dithionite pH 7.4) was exposed to air.
This resulted in visible red/orange
precipitation.
2.2.7 Oligonucleotide probes
The following oligonucleotides were purchased from either Operon Biotechnologies Inc.
(Alameda, CA) or Integrated DNA Technologies, Inc. (Coralville, IA), in their HPLC and
PAGE purified form: (-RARE-16) – CAACCGAAAGTTCACTC and its complement;
nonspecific sequence – TGTTTCTGCCTCTGT and its complement. One strand of each
oligonucleotide pair was 5’ end-labeled with fluorescein (Figure 2.5). Upon receipt, the
oligonucleotides were resuspended in DNAse-free water and quantified. The
complementary oligonucleotides were then mixed at 1.25:1 unlabeled:labeled ratio in 10
mM Tris (pH 8.0) and 10 mM NaCl annealing buffer. The annealing reaction was placed
in a water bath set to 10°C higher than the melting temperatures of the complementary
oligonucleotides. The water bath was then turned off and the annealing reaction allowed
43
to cool overnight. The resultant double-stranded oligonucleotides were quantified and
stored at -20°C.
2.2.8 Fluorescence Anisotropy
A fluorescence anisotropy (FA) assay was utilized to quantify NZF-1-F2F3/DNA
binding. Experiments were performed on an ISS PC-1 spectrofluorimeter configured in
the L format. Initially, a full excitation/emission spectrum was measured to determine the
optimum excitation/emission wavelengths for the experiment. A wavelength/band pass of
493 nm/2 nm for excitation and 517 nm/1 nm for emission were utilized for the
experiments. All binding reactions were carried out using a 10 nM fluorescently labeled
DNA solution containing 0.05 mg/mL bovine serum albumin (BSA), in a Spectrosil farUV quartz window fluorescence cuvette (Starna Cells). BSA was included to prevent
adherence of either the protein or the DNA to the cuvette walls. Titrations with the
Zn(II)- NZF-1-F2F3, Fe(II)-NZF-1-F2F3 and apo-NZF-1-F2F3 were carried out in 100
mM Tris, 100 mM NaCl at pH 7.4. 150 μM sodium diothionite was included in the
Fe(II)-NZF-1-F2F3 experiments. In a typical titration, the peptide was added
incrementally to the cuvette containing the DNA and the anisotropy, r, was monitored
until saturation was reached. Each fluorescence anisotropy data point represents the
average of 60 readings taken over a period of 115 seconds, and titrations were carried out
in triplicate. Anisotropy, r, was converted to fraction bound, Fbound (fraction of peptidebound DNA at a given DNA concentration), according to the following equation (27):
Fbound 
r  r free
(rbound  r )Q  (r  r free )
44
where rfree is the anisotropy of the fluorescein-labeled oligonucleotide and rbound is the
anisotropy of the peptide-DNA complex at saturation. Q is the quantum yield and is
applied as a correction factor to account for changes in fluorescence intensity over the
course of the experiment (Q = Ibound/Ifree) (27). Typically, Q was 0.95. Fbound was then
plotted against peptide concentration, and the data fit to a one-site binding model:
P+D
Kd 
Fbound 
PD
[ P][ D]
[ PD ]
Ptotal  Dtotal  K d  ( Ptotal  Dtotal  K d ) 2  4 Ptotal Dtotal
2 Dtotal
where P is the protein concentration and D is the DNA concentration. A control
experiment in which Fe(II) cations were titrated with -RARE -16 DNA under the same
experimental conditions as the protein titration was also performed. No changes in
anisotropy were observed.
2.3 Results
2.3.1 Peptide Preparation
NZF-1 and its homologs contain clusters of zinc finger domains (Figure 2.1), and several
groups have over-expressed and purified peptides that contain one to five of these
domains (2, 3, 11, 19, 28). We prepared a peptide fragment of NZF-1 called NZF-1-F2F3
that contains zinc fingers 2 and 3. This fragment is the minimal fragment that has been
shown to induce DNA binding when zinc was bound (11, 17). A forward titration with
cobalt and a competitive titration with zinc confirmed that these metal ions bound to the
45
construct we had prepared. The cobalt bound species exhibited d-d transitions at
wavelengths diagnostic of tetrahedral coordination geometry (11).
2.3.2 UV-visible spectrum of Fe(II)-NZF-1-F2F3
The addition of two equivalents of (NH4)2Fe(SO4)2 to NZF-1-F2F3 in the presence of 150
M sodium dithionite resulted in the appearance of absorption bands around 312 and 347
nm (Figure 2.3). We propose that these are charge transfer bands based upon their
similarity to bands observed for iron(II) substituted TTP-2D (a CCCH type zinc finger
protein) (290, 338 nm); iron(II) substituted consensus peptide which is a model zinc
finger peptide (280, 340 nm); and tetrathiolate peptide/protein sites (310 and 333 nm)
(22, 29-31).
2.3.3 Addition of Co(II) to Fe(II)-NZF-1-F2F3
Stoichiometric Co(II) was titrated into solutions of Fe(II)-NZF-1-F2F3 to determine if
0.2
absorbance
0.15
0.1
0.05
0
300
400
500
600
700
800
wavelength (nm)
Figure 2.3 Absorption spectrum of Fe(II)-NZF-1-F2F3. Spectra recorded in 200 mM
HEPES, 100 mM NaCl, 150 M Na3O4S4 buffer at pH 7.4.
46
1
absorbance
0.8
Co(II) and Fe(II)
CT bands
0.6
octahedral
Co(II)
0.4
tetrahedral
Co(II)
0.2
0
300
400
500
600
700
800
wavelength (nm)
Figure 2.4 UV visible spectrum of two equivalents of Co(II) added into 50 µM Fe(II)NZF-1-F2F3. Bands for tetrahedral and octahedral cobalt species are observed as well as
charge transfer bands for both Co(II)-NZF-1-F2F3 and Fe(II)-NZF-1-F2F3.
cobalt could replace iron. Distinct d-d transitions between 520-720 nm indicative of
Co(II) binding appeared and the shape and intensity of the peaks between 240-400 nm
changed suggesting that cobalt(II) had replaced iron(II) within the metal binding site [19]
(Figure 2.4). At a 1:1 ratio of Fe(II) to Co(II), a band around 400 nm indicative of
octahedral cobalt was also present, suggesting that not all of the Co(II) had replaced the
iron (19). The Co(II) substitution experiments are suggestive that Fe(II) binds at the same
Cys2HisCys site on NZF-1 as Co(II) and by inference, Zn(II) [19].
2.3.4 Air Oxidation of Fe(II)-NZF-1-F2F3
A crude oxidation of Fe(II)-NZF-1-F2F3 was accomplished by exposing anaerobic
Fe(II)-NZF-1-F2F3 to air. Visible precipitation of a red/orange solid occurred. This
precipitation suggests oxidation of Fe(II), followed by hydrolysis of the ferric-NZF-147
Figure 2.5 Sequences of the fluorescein labeled -RARE and nonspecific probes
used for fluorescence anisotropy studies. The AGTT core recognition sequence is
boxed.
F2F3 to complex iron-oxide species, which is a common phenomenon for aerobic ferric
complexes (32).
2.3.5 DNA Binding of M-NZF-1-F2F3 to  -RARE DNA
There have been several studies examining the target DNA sequences for both NZF-1
and MyT1. Both proteins recognize binding sites containing a core AGTT sequence. For
high affinity binding to be observed, at least two zinc finger domains are required (19).
The current literature reports dissociation constants (Kd) for DNA binding that range
from micromolar to nanomolar for NZF-1 or MyT1(11, 19).
These affinities were
determined by using a variety of constructs (e.g. two zinc fingers, three zinc fingers, with
and without affinity tags) and different techniques (e.g. DNase I footprinting, surface
48
Figure 2.6 Change in anisotropy (as fraction bound) upon the addition of Zn(II)NZF-1-F2F3 to -RARE DNA. (buffer: 100 mM Tris, 100 mM NaCl at pH 7.4).
plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy) (11, 19,
33). The variation in constructs studied and technique used likely accounts for the
disparities in reported DNA binding affinities.
Here we used the technique of
fluorescence anisotropy (FA) to study NZF-1-F2F3/DNA binding. This technique is
solution based and gives a true equilibrium value for binding, allowing one to rapidly and
systematically study the interactions between macromolecules (27). We used FA to
compare the Zn(II)-NZF-1-F2F3/DNA binding interaction with that of the Fe(II) and apo
forms of NZF-1-F2F3 to ascertain whether DNA binding requires specific metal ions and
understand the specificity. For this assay M-NZF-1-F2F3 (M = Zn(II), Fe(II), apo) was
titrated with fluorescently labeled double stranded DNA and the change in anisotropy
was monitored (27). Double stranded oligonucleotides corresponding to the -RARE
recognition sequence (-RARE-16) and a nonspecific sequence (Figure 2.5) were used
for these studies. These fluorescein-labeled DNA fragments had previously been
49
Figure 2.7 Change in anisotropy (as fraction bound) upon the addition of Fe(II)-NZF1-F2F3 to -RARE DNA. (buffer: 100 mM Tris, 100 mM NaCl, 150 μM sodium
diothionite at pH 7.4).
demonstrated to be amenable to FA by Davis and Berg and we followed a slightly
modified protocol for our studies (33).
Using FA, dissociation constants of 11  2 nM and 12  2 nM were found for binding
of the Zn-NZF-1-F2F3 and Fe(II)-NZF-1-F2F3 peptides with -RARE oligonucleotide,
respectively (Figures 2.6 and 2.7). Neither of these metal-peptide complexes exhibited
any affinity for the random DNA sequence of the same length leading us to conclude that
the DNA recognition was sequence specific. Apo-NZF-1-F2F3 titrations could not be
completed due to precipitation of the peptide. This data and previous 1H NMR data
indicating that the metal-free peptide was in an unfolded state [10] confirm that the
unfolded peptide is incapable of DNA binding. The ability of Fe(II)-NZF-1-F2F3 to
selectively recognize a physiologically relevant DNA sequence and bind with the same
affinity as the zinc bound form of the protein implies that it can be a functional substitute
in vitro.
50
2.4 Discussion
NZF-1 is a member of a class of non-classical zinc finger proteins that contain
multiple domains with a Cys-X4-Cys-X4-His-X7-His-X5-Cys sequence motif (11, 17).
Two homologs of NZF-1 are known: MyT1 (NZF-2) and NZF-3 (or ST18) (2,3, 5, 11,
17, 19, 34). The sequence identity between individual domains within a given protein is
high (60-100%). The domain clusters also demonstrate a high degree of identity and
homology between the different proteins inclusive of the linker regions between the
domains. Structural analysis of a single finger domain of NZF-1 using multidimensional
NMR spectroscopy revealed a CCHC metal binding motif (17), although we refer to them
as CCHHC domains due to the presence of the additional conserved histidine and to
distinguish them from other CCHC zinc fingers. The zinc binding domains bind DNA in
a modular fashion requiring a minimum of two zinc domains for appreciable affinity, a
property that is commonly observed for zinc finger proteins (2, 3, 11).
Metal coordination to the cysteine and histidine ligands of the NZF-1/MyT1 type
domains is necessary for DNA binding: when zinc is removed from MyT1 by addition of
the chelator 1,10-orthophenantroline the protein does not bind DNA (3). Preliminary
results also indicated that high concentrations of cadmium(II) and copper(II) displace
zinc and abolish the DNA binding activity of the MyT1 domains, while cobalt(II),
manganese(II) and magnesium(II) showed no effect [2]. Given the importance of NZF-1
and MyT1 in neuronal development, and the evidence that iron overload is associated
with deleterious neuronal events, we sought to determine if iron could coordinate to NZF1 and to understand the consequences of this metal replacement on the function of these
zinc binding domains.
51
We found that ferrous iron could coordinate to a two domain fragment of NZF-1
representing a minimal DNA-binding unit and that these complexes were stable when
kept under strictly anaerobic conditions.
Ferrous iron coordination resulted in the
appearance of bands in the UV-region of the electronic spectrum similar to those reported
for CP-1 (a consensus zinc finger peptide) (22) and TTP-2D (a zinc finger protein
involved in inflammation) (29) and which we propose are charge transfer bands. Cobalt
substitution experiments in which two equivalents of Co(II) were added to solutions of
Fe(II)-NZF-1-F2F3 resulted in the appearance of Co(II) d-d bands and changes in the
charge transfer bands. The Co(II) d-d bands between 520-720 were indicative of
tetrahedral geometry and look as expected for the Co(II)-NZF-1-F2F3 complex [10]. The
band at 400 nm is diagnostic of Co(II) octahedral geometry and suggests the presence of
some unbound cobalt. On the basis of these findings, we propose that iron coordinates to
the same site as cobalt and, by inference zinc.
Since the identification of the NZF-1/MyT1 protein family, a number of studies
have reported on the DNA binding properties of the CCHHC zinc binding domains. Kim
and Hudson used electrophoretic mobility shift assays (EMSA) on bacterial extracts to
determine that a two finger construct of MyT1 can bind to the pit-1 promoter element (3).
Jiang and co-workers used EMSA with GST-fusions of NZF-1 to determine that at least
two zinc fingers from NZF-1 must be present for appreciable binding to the -RARE
promoter element and that a three finger construct shows higher DNA sequence
specificity than the two finger construct (2). Competition experiments within this study
identified a core AGTT unit necessary for NZF-1 binding to -RARE. Through DNAse I
footprinting, a dissociation constant of 1.6 M was calculated for the NZF-1/-RARE
52
interaction (11). More recently, Mackay and co-workers used a surface plasmon
resonance/isothermal titration calorimetry approach to measure dissociation constants for
binding of the individual zinc fingers of MyT1 to -RARE (19). Under low salt
concentrations, mid-micromolar Kd’s were observed for three of the seven domains,
whereas a two domain construct exhibited DNA binding in the high nanomolar range
(270 nM). Additionally, Davis and Berg used fluorescence anisotropy to measure
dissociation constants of a two domain fragment of NZF-1 (as well as two mutants of this
peptide) as a function of salt concentration (33). They observed mid-nanomolar Kd’s. The
discrepancies in binding affinities between these studies is likely attributable to the
variety of techniques, experimental conditions and protein constructs utilized.
Fluorescence anisotropy is an extremely versatile technique sensitive to changes in
protein and DNA size as well as the microenvironment (e.g. level of salt or pH) and NZF1 fragments were known to be amenable to this technique (33). Thus, we sought to use
this technique to systematically survey the DNA binding properties of NZF-1 as a
function of metal ion bound.
FA titrations of M-NZF-1-F2F3 [M = Zn(II), Fe(II) or apo] with -RARE-16 as a
function of metal ion bound revealed that metal coordination was required for DNA
binding but that the identity of the metal ion was flexible. Similar dissociation constants
(~ 11 nM) were observed when either zinc or ferrous iron was coordinated to NZF-1F2F3. In order for zinc finger proteins to bind to their target DNA sequences, they must
be folded into a conformation that is favorable for DNA recognition. Because Fe(II)NZF-1-F2F3 binds DNA with the same affinity and selectivity as Zn(II)-NZF-1-F2F3, we
can infer that Fe(II) promotes a similar fold as Zn(II). In contrast, apo-NZF-1-F2F3 is
53
incapable of binding to its target DNA, and instead precipitates under buffered conditions
implying that the absence of metal prevents proper folding.
The measured dissociation constants for -RARE DNA binding to both Zn(II)NZF-1-F2F3 and Fe(II)-NZF-1-F2F3 that we report here are significantly lower than
those determined for -RARE DNA binding to Zn(II)-NZF-1-F2F3 by DNaseI (10). It
can be difficult to measure reliable affinities using gel-based methods due to the ‘caging
effect’ of the gel matrix and the solution based fluorescence anisotropy experiment likely
provides a more accurate binding constant. The affinity of a two domain construct of
MyT1 with a comparable DNA sequence was reported to be approximately 20-times
larger than the dissociation constants we reported here for NZF-1-F2F3, even though
another reliable solution based method, isothermal titration calorimetry was used. (19)
While MyT1 is able to bind the -RARE binding site, this site is not a recognized in vivo
target for this protein explaining why weaker binding was measured.
NZF-1 is found primarily in the brain where it is localized to motor and sensory
neurons. Iron is vital for a number of metabolic processes in the brain, including neuronal
development, myelin formation and the synthesis and metabolism of neurotransmitters
(35). However, the levels of iron in the brain must be tightly regulated in part because
iron is redox active and can promote oxidative damage. Accumulation of brain iron has
been linked to neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases
(26). One of the hallmarks of these diseases is iron-mediated oxidative stress.
Additionally, certain types of nerve cells, including neurons which are associated with
expression of NZF-1 and MyT1, are extremely sensitive to iron overload and exhibit
decreased cell viability (36). The vulnerability of nerve cells to the misregulation and
54
reactivity of iron leads us to hypothesize that binding of excess iron from iron overload to
NZF-1 would be a negative event: the ability of the iron-bound protein to maintain its
DNA binding properties could lead to oxidative damage of the DNA, with serious
consequences to the cell. Given the thermodynamic preference for zinc, the increased
levels of iron in the cell will not likely result in all coordinated zinc being replaced by
iron, but even a fraction of iron coordination has the potential to be deleterious.
The studies presented here establish, for the first time, that iron can bind to a
functional fragment of NZF-1 without disrupting its DNA binding ability. The
importance of NZF-1 for proper neuronal development coupled with a well established
but little understood role of iron in a number of neurodegenerative diseases make further
investigations of iron substituted NZF-1 of interest. Studies to address the potential for
iron-bound NZF-1 to cause DNA damage are in progress.
55
2.5 References
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Functional characterization of iron-substituted neural zinc finger factor 1: metal
and DNA binding, Journal of biological inorganic chemistry : JBIC : a
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2.
Jiang, Y., Yu, V. C., Buchholz, F., O'Connell, S., Rhodes, S. J., Candeloro, C.,
Xia, Y. R., Lusis, A. J., and Rosenfeld, M. G. (1996) A novel family of Cys-Cys,
His-Cys zinc finger transcription factors expressed in developing nervous system
and pituitary gland, J Biol Chem 271, 10723-10730.
3.
Kim, J. G., and Hudson, L. D. (1992) Novel member of the zinc finger
superfamily: A C2-HC finger that recognizes a glia-specific gene, Molecular and
cellular biology 12, 5632-5639.
4.
Bellefroid, E. J., Bourguignon, C., Hollemann, T., Ma, Q., Anderson, D. J.,
Kintner, C., and Pieler, T. (1996) X-MyT1, a Xenopus C2HC-type zinc finger
protein with a regulatory function in neuronal differentiation, Cell 87, 1191-1202.
5.
Kim, J. G., Armstrong, R. C., v Agoston, D., Robinsky, A., Wiese, C., Nagle, J.,
and Hudson, L. D. (1997) Myelin transcription factor 1 (Myt1) of the
oligodendrocyte lineage, along with a closely related CCHC zinc finger, is
expressed in developing neurons in the mammalian central nervous system, J
Neurosci Res 50, 272-290.
6.
Yee, K. S., and Yu, V. C. (1998) Isolation and characterization of a novel member
of the neural zinc finger factor/myelin transcription factor family with
transcriptional repression activity, J Biol Chem 273, 5366-5374.
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7.
Weiner, J. A., and Chun, J. (1997) Png-1, a nervous system-specific zinc finger
gene, identifies regions containing postmitotic neurons during mammalian
embryonic development, The Journal of comparative neurology 381, 130-142.
8.
Gu, G., Wells, J. M., Dombkowski, D., Preffer, F., Aronow, B., and Melton, D. A.
(2004) Global expression analysis of gene regulatory pathways during endocrine
pancreatic development, Development 131, 165-179.
9.
Matsushita, F., Kameyama, T., and Marunouchi, T. (2002) NZF-2b is a novel
predominant form of mouse NZF-2/MyT1, expressed in differentiated neurons
especially at higher levels in newly generated ones, Mechanisms of development
118, 209-213.
10.
Nielsen, J. A., Berndt, J. A., Hudson, L. D., and Armstrong, R. C. (2004) Myelin
transcription factor 1 (Myt1) modulates the proliferation and differentiation of
oligodendrocyte lineage cells, Mol Cell Neurosci 25, 111-123.
11.
Berkovits, H. J., and Berg, J. M. (1999) Metal and DNA binding properties of a
two-domain fragment of neural zinc finger factor 1, a CCHC-type zinc binding
protein, Biochemistry 38, 16826-16830.
12.
Berg, J. M., and Shi, Y. (1996) The galvanization of biology: a growing
appreciation for the roles of zinc, Science (New York, N.Y 271, 1081-1085.
13.
Laity, J. H., Lee, B. M., and Wright, P. E. (2001) Zinc finger proteins: new
insights into structural and functional diversity, Current opinion in structural
biology 11, 39-46.
14.
Maret, W. (2004) Zinc and sulfur: a critical biological partnership, Biochemistry
43, 3301-3309.
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15.
Matthews, J. M., and Sunde, M. (2002) Zinc fingers--folds for many occasions,
IUBMB life 54, 351-355.
16.
Berg, J. M., and Godwin, H. A. (1997) Lessons from zinc-binding peptides,
Annual review of biophysics and biomolecular structure 26, 357-371.
17.
Berkovits-Cymet, H. J., Amann, B. T., and Berg, J. M. (2004) Solution structure
of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA
binding, Biochemistry 43, 898-903.
18.
Suetake, T., Nagashima, T., Hayashi, F., and Yokoyama, S. (2006) Solution
structure of tandem repeat of the fifth and sixth zinc-finger C2HC domains from
human ST18, PDB pdb2cs8.
19.
Gamsjaeger, R., Swanton, M. K., Kobus, F. J., Lehtomaki, E., Lowry, J. A.,
Kwan, A. H., Matthews, J. M., and Mackay, J. P. (2008) Structural and
biophysical analysis of the DNA binding properties of myelin transcription factor
1, J Biol Chem 283, 5158-5167.
20.
Berg, J. M., and Merkle, D. L. (1989) On the metal ion specificity of zinc finger
proteins J Am Chem Soc 111, 3759-3761.
21.
Conte, D., Narindrasorasak, S., and Sarkar, B. (1996) In vivo and in vitro ironreplaced zinc finger generates free radicals and causes DNA damage, The Journal
of biological chemistry 271, 5125-5130.
22.
diTargiani, R. C., Lee, S. J., Wassink, S., and Michel, S. L. (2006) Functional
characterization of iron-substituted tristetraprolin-2D (TTP-2D, NUP475-2D):
RNA binding affinity and selectivity, Biochemistry 45, 13641-13649.
58
23.
Hanas, J. S., Hazuda, D. J., Bogenhagen, D. F., Wu, F. Y., and Wu, C. W. (1983)
Xenopus transcription factor A requires zinc for binding to the 5 S RNA gene,
The Journal of biological chemistry 258, 14120-14125.
24.
Omichinski, J. G., Trainor, C., Evans, T., Gronenborn, A. M., Clore, G. M., and
Felsenfeld, G. (1993) A small single-"finger" peptide from the erythroid
transcription factor GATA-1 binds specifically to DNA as a zinc or iron complex,
Proc Natl Acad Sci U S A 90, 1676-1680.
25.
Connor, J. R., and Menzies, S. L. (1996) Relationship of iron to oligodendrocytes
and myelination, Glia 17, 83-93.
26.
Connor, J. R., and Menzies, S. L. (1995) Cellular management of iron in the
brain, J Neurol Sci 134 Suppl, 33-44.
27.
Lakowicz, J. (1999) Principles of Fluorescence Spectroscopy, 2nd ed., Kluwer
Academic/Plenum Publishers, New York.
28.
Blasie, C. A., and Berg, J. M. (2000) Toward ligand identification within a
CCHHC zinc-binding domain from the NZF/MyT1 family, Inorganic chemistry
39, 348-351.
29.
Krizek, B. A., and Berg, J. M. (1992) Complexes of Zinc Finger Peptides with
Ni2+ and Fe2+, Inorganic chemistry 31, 2984-2986.
30.
Lombardi, A., Marasco, D., Maglio, O., Di Costanzo, L., Nastri, F., and Pavone,
V. (2000) Miniaturized metalloproteins: application to iron-sulfur proteins, Proc
Natl Acad Sci U S A 97, 11922-11927.
31.
Lovenberg, W., and Sobel, B. E. (1965) Rubredoxin: A New Electron Transfer
Protein from Clostridium pasteruianum, Proc Natl Acad Sci U S A 54, 193-199.
59
32.
Cotton, A. F., and Wilkinson, G. (1988) Advanced Inorganic Chemistry.
33.
Davis, A. M. (2006) Biophysical characterization of zinc(II)-binding domains in
two systems: Neural Zinc Finger Factor-1 and T-cell co-receptors CD4 and CD8
alpha with T-Cell-specific kinase Lck, In Department of Biophysics and
Biophysical Chemistry, p 143, Johns Hopkins University School of Medicine,
Baltimore.
34.
Jandrig, B., Seitz, S., Hinzmann, B., Arnold, W., Micheel, B., Koelble, K.,
Siebert, R., Schwartz, A., Ruecker, K., Schlag, P. M., Scherneck, S., and
Rosenthal, A. (2004) ST18 is a breast cancer tumor suppressor gene at human
chromosome 8q11.2, Oncogene 23, 9295-9302.
35.
Gaasch, J. A., Lockman, P. R., Geldenhuys, W. J., Allen, D. D., and Van der
Schyf, C. J. (2007) Brain iron toxicity: differential responses of astrocytes,
neurons, and endothelial cells, Neurochem Res 32, 1196-1208.
36.
Kress, G. J., Dineley, K. E., and Reynolds, I. J. (2002) The relationship between
intracellular free iron and cell injury in cultured neurons, astrocytes, and
oligodendrocytes, J Neurosci 22, 5848-5855.
60
Chapter 3
Switching Metal Ion Coordination and DNA Recognition in a Tandem
CCHHC-type Zinc Finger Peptide*
3.1 Introduction
Zinc fingers (ZFs) are proteins which contain modular domains that coordinate
zinc ions via a combination of four cysteine and/or histidine ligands to fold into specific
three- dimensional structures (1-10). ZFs have critically important biological roles in
modulating transcription and translation and show a remarkable capacity for highly
selective nucleic acid recognition (3, 4, 10). Zinc proteins are ubiquitous in eukaryotes:
approximately 10% of the human genome encodes for these proteins (5, 8, 11-13). ZF
proteins can be separated into at least 14 different classes which are distinguished by the
ligand set involved in Zn(II) coordination and, when known, the three dimensional
structure of the folded form. The best-studied class of ZFs are the ‘classical’ ZFs (5, 9,
14). Classical ZFs utilize a Cys2His2 (CCHH) ligand set to coordinate zinc ions and fold
into a recognizable alpha helix/beta sheet structure upon zinc ion coordination (1-10).
The remaining classes of ZFs are often referred to as ‘non-classical’ ZFs.(1) Nonclassical ZFs are known to be involved in key biological processes including mRNA
processing, viral replication, tumor suppression and neuronal development (1, 15-19);
yet, in many instances little is known about their biochemical roles and consequently, the
* Adapted from the publication: Besold, A.N.; Oluyadi, A.A.; Michel, S.L.J. 2013. Inorg.
Chem, 52(8):4721-8.
61
biophysical basis of nucleic acid recognition for these non-classical ZF proteins is
unresolved.
One important class of non-classical ZFs are the Cys2His2Cys or CCHHC ZFs (1).
This is a small class of ZFs, with only three homologs identified to date: Neural Zinc
Finger Factor-1 (NZF-1), Myelin Transcription Factor 1 (MyT1) and Suppression of
Tumorigenicity 18 (ST18) (18-31). NZF-1 and MyT1 are found in the Central Nervous
System (CNS) where they play crucial roles in development: misregulation of either
protein is associated with schizophrenia, mental retardation, brain cancer, and
periventricular leukomalacia (a common cause of cerebral palsy) (21, 29, 30, 32-41).
NZF-1 is found in neurons where it regulates β-retinoic acid receptor (β-RAR)
expression, while MyT1 is found in oligodendrocytes, where it regulates expression of
the proteolipid protein (PLP), the main myelin forming protein in the CNS, as well as
2’3’-cyclic-nucleotide 3’phosphodiesterase (CNP) and opalin (26, 29, 30, 42).
Both NZF-1 and MyT1 contain multiple CCHHC ZF domains, which are
arranged in clusters of 1, 2, 3 or 4 ZFs (Figure 3.1a) (1, 29, 30). The amino acid
sequences of each ZF domain within either NZF-1 or MyT1 are remarkably similar, with
upwards of 100% sequence identity (Figure 3.1b). This is unusual for a ZF protein.
Typically, amino acid sequence similarity for homologous ZF domains is more limited
and restricted to the coordinating ligands and a few additional amino acids that are
involved in stabilizing the protein fold or promoting nucleic acid recognition (8, 10).
The presence of five potential metal binding ligands (CCHHC) in the ZF
sequences of NZF-1 and MyT1 suggests a possible five-coordinate geometry at the
zinc(II) sites (43); however, structural and optical data for singular and double ZF
62
(a)
(b)
rNZF-1-F2 (488-529)
D V K K Y Y D P S R T E K R E S K C P T P G C D G T G H V T G L Y P H H R S L S G C
rMyT1 -F2 (222-263)
K S C Y N K D P S R V E K R E I K C P T P G C D G T G H V T G L Y P H H R S L S G C
mMYT1 -F4 (792-836)
S K D I K K E L L T C P T P G C D G S G H I T G N Y A S H R S L S G C
rNZF-1-F3 (530-594) P H K D R V P P E I L A M H E N V L K C P T P G C T G R G H V N S N R N S H R S L S G C P I A A A E K L A K A
rMyT1 -F3 (264-318) P H K D R I P P E I L A M H E N V L K C P T P G C T G Q G H V N S N R N T H R S L S G C P I A A A E K L A K S
mMYT1 -F5 (837-880) P L A D K S L R N L M A A H S A D L K C P T P G C D G S G H I T G N Y A S H R S L S G C R A K K S G L R V
(c)
(d)
Y520
Y861
H858
H515
X557
X854
Figure 3.1 Alignment and Structures of NZF-1 and MyT1 (a) Cartoon diagram of the ZF
topology of NZF-1 and MyT1 from Rattus norvegicus, r or Mus Musculus, m. Individual ZF
domains are boxed and the alignment shows the ZF clustering. Note, F1 is absent in rMyT1.
(b) Alignment of the amino acid sequences of NZF1-F2F3, MyT1-F2F3, and MyT1-F4F5.
The cysteine and histidine ligands that can directly coordinate Co(II) and Zn(II) are highlighted
in blue, the amino acids that have been proposed to participate in a stacking interaction with
the non-coordinating histidine are highlighted in red, and the amino acids that differ between
the individual ZF domains of NZF-1 and MyT1 are highlighted in green. (c) The NMR
solution structure of F2 of rNZF-1 (PDBID IPXE). The highlighted amino acids are color
coded to match those that are highlighted in Figure 3.1b. Amino acid position shown to be
important for NZF-1-F2F3 DNA recognition shown in green. (d) NMR solution structure of
finger 5 mMyT1 (PDBID 2JYD). Highlighted amino acids are color coded to match those in
figure 3.1b. The amino acid position that has been shown to be important for NZF-1-F2F3
DNA recognition highlighted in green. The structural figures were generated in Pymol.
domains of both NZF-1 and MyT1 all indicate four coordinate geometry at the Zn(II)
sites (18, 19, 25, 27). The structural data for NZF-1 and MyT1 are limited to NMR
structures of singular domains (F2 for NZF-1 and F5 for MyT1) (Figure 3.1c and 3.1d)
(19, 27). In both structures, Zn(II) is coordinated to three cysteine residues and one
histidine residue in a tetrahedral geometry (19, 27). The coordinating histidine is the
63
second conserved histidine in the sequence. The first histidine has been proposed to
participate in a stacking interaction with a highly conserved tyrosine, stabilizing the
structure (19). Interestingly, when the second histidine is mutated to a non-coordinating
alanine or glutamine, for either F2 or F3 of NZF-1, Zn(II) coordination is still observed,
indicating that the coordinating ligands are flexible (19, 25). The effect of this alternate
coordination on function is not known.
A bona fide DNA target sequence has only been identified for a two ZF domain
(F2+F3) of NZF-1 (18).
This target sequence is from the β-RAR promoter and it
contains an AAGTT sequence that has been proposed to be a general recognition
sequence for all CCHHC-type ZFs (18, 24, 29). However, a two ZF domain construct of
MyT1 exhibits significantly weaker affinity for β-RAR DNA when compared to the
affinity measured for NZF-1 (F2+F3) (27, 28). This suggests that MyT1 recognizes a
different DNA sequence than NZF-1. In addition, the promoter sequences of the genes
that are recognized by MyT1 do not always contain the AAGTT sequence, consistent
with the idea that MyT1 binds to a different DNA recognition sequence than NZF-1 (42).
From these studies, two important questions have emerged: (1) what is the
functional role of the non-coordinating histidine that is present in all CCHHC type ZF
proteins? and (2) How do NZF-1 and MyT1 discriminate between DNA targets, given the
high sequence similarity between individual ZFs? By preparing mutations of the two-ZFdomain peptide construct of NZF-1 (F2+F3), for which a bona fide DNA target sequence
has been identified, and of the analogous two-ZF domain construct of MyT1 (F2+F3), we
have discovered that the non-coordinating histidine is critical for DNA recognition. We
also report that a singular residue that is not conserved between the NZF-1-F2F3 and
64
MyT1-F2F3 sequences that is responsible for ‘high affinity’ (nanomolar) DNA binding.
These results allow us to propose a unique paradigm of zinc ion mediated DNA
recognition for this novel class of non-classical ZF proteins.
3.2 Materials and Methods
3.2.1 Nomenclature
All peptide constructs of NZF-1 and MyT1 are named based upon the ZFs included
within their sequences. For example, the construct that contains the second (F2) and third
(F3) ZF of NZF-1 from Rattus norvegicus (R. norvegicus) is named NZF-1-F2F3. Mutant
peptides in which either the first (H515 and H559) or second (H523 and H567) histidine
have been mutated to a phenylalanine are referred to as CCFHC or CCHFC, respectively.
3.2.2 Expression and Purification of NZF-1-F2F3 and MyT1-F2F3
A DNA fragment corresponding to residues 487-584 of full length NZF-1 from R.
norvegicus, termed NZF-1-F2F3 ligated into a pET15b vector in which the hexahistidine
tag had been removed was a generous gift of Dr. Holly Cymet (Stevenson University).
The DNA corresponding to residues 222-318 of full length MyT1, termed MyT1-F2F3,
was amplified via the Polymerase Chain Reaction (PCR) from a R. norvegicus brain
cDNA library, which was a generous gift from Dr. Anthony Lanahan (Yale University).
This DNA fragment was ligated into a pET15b vector using the restriction sites NcoI and
BamHI so that the hexahistidine tag was removed. To express these constructs, the
vectors were transformed into BL21(DE3) Escherichia coli (Novagen) cells and the cells
65
were then grown in Luria Bertani (LB) broth with 100 µg/mL ampicillin at 37°C until
mid-log phase. Protein expression was induced with 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG) and grown for 4 hours post induction. Cells were harvested
by centrifugation at 7800 x g for 15 minutes at 4°C. Cell pellets were resuspended in 25
mM Tris [tris(hydroxymethyl)aminomethane] at pH 8.0, 100 µM ZnCl2, and 5 mM
dithiothreitol (DTT) containing a mini ethylenediaminetetraacetic acid (EDTA) free
protease inhibitor tablet (Roche) and lysed via sonication on a Misonix Sonicator 3000.
Cell debris were removed by centrifugation at 12100 x g for 15 minutes at 4°C. The
bacterial supernatant was loaded onto a SP Sepharose Fast Flow column (Sigma) and the
protein was eluted with a stepwise salt gradient from 0 to 1 M KCl. The cysteine thiols of
the protein were reduced by incubation with 10 mM tris(2-carboxyethyl)phosphine
(TCEP) (Thermo) at room temperature for 30 minutes. The protein was further purified
via High Performance Liquid Chromatography (HPLC) using a Waters 626 LC system
and a Waters Symmetry Prep 300 C18 7 µm reverse phase column with an acetonitrile
gradient containing 0.1% trifluoroacetic acid (TFA). The proteins eluted at 29%
acetonitrile with 0.1% TFA. The collected proteins were dried using a Thermo Savant
SpeedVac concentrator housed in a Coy anaerobic chamber (97% N2/3% H2).
3.2.3 Design of NZF-1-F2F3 and MyT1-F2F3 Mutants
Mutations of either the first or second conserved histidine residue of NZF-1-F2F3 to
phenylalanine were made using a Quikchange Mutagenesis Kit (Agilent). To create the
CCFHC mutant, H515 and H559, which correspond to the first conserved histidine in
each ZF, were mutated to a phenylalanine. Similarly, H523 and H567, which correspond
66
to the second conserved histidine in each ZF, were mutated to a phenylalanine to create
the CCHFC mutant. A remaining non-conserved histidine residue (amino acid 522) was
mutated to an alanine to prevent any adventitious metal ion coordination. Glutamine 291
of MyT1-F2F3 was mutated to an arginine using the Quikchange Mutagenesis Kit in
order to create the Q291R MyT1-F2F3 mutant. All mutations were confirmed using DNA
sequencing at the Biopolymer/Genomics Core Facility housed at the University of
Maryland School of Medicine.
Expression and purification followed the procedure
previously described (vide infra).
3.2.4 Metal Binding Studies
Metal ion titrations were performed on a PerkinElmer Lambda 25 UV-visible
Spectrometer, CoCl2 and ZnCl2 were from Fisher Scientific. In a typical experiment, 20 50 µM apo-peptide was titrated with CoCl2 and the absorbance was monitored until
saturation. The relative affinity of the peptides for Zn(II) were determined by monitoring
the displacement of Co(II) by Zn(II) following the method developed by Berg and
Merkle.(14) All titrations were performed in 200 mM HEPES [4-(2-hydroxyethyl)-1piperazineethanesulfonic acid], 100 mM NaCl at pH 7.5. Data were fit to an appropriate
binding equilibria using linear least squares analysis (KaleidaGraph, Synergy Software).
3.2.5 Circular Dichroism
Far-UV Circular Dichroism (CD) was performed on a Jasco-810 Spectropolarimeter. 20
µM of apo-peptide was prepared in 300 µL of 25 mM Sodium Phosphate, pH 7.5. Molar
equivalents of either CoCl2 or ZnCl2 were added to the apo-peptide and the spectra were
67
measured. All spectra were collected from 190 nm to 260 nm, with a scan rate of 100
nm/min, at 25°C in a 1 mm path length quartz rectangular cell (Starna Cells). A total of 5
scans were obtained for each point and the average was displayed.
3.2.6 Oligonucleotide Probes
Oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville,
IA), in their HPLC purified form. The -RAR DNA, CACCGAAAGTTCACTC, was
purchased with a 5’ end-labeled fluorescein along with its unlabeled complement. A
random DNA strand, TGTTTCTGCCTCTGT, was also purchased with a 5’ end-labeled
fluorescein along with its unlabeled complement. The oligonucleotides were annealed by
mixing a ratio of 1.25:1 unlabeled:labeled in 10 mM Tris, pH 8.0 and 10 mM NaCl
annealing buffer. The annealing mixture was placed in a water bath set to 10°C higher
than the melting temperature of the DNA strands. The annealing reaction proceeded for 5
minutes before the water bath was turned off and allowed to cool overnight. The resultant
double-stranded oligonucleotides were quantified and stored at -20°C.
3.2.7 Fluorescence Anisotropy
Fluorescence anisotropy (FA) assays were performed on an ISS PC-1 spectrofluorimeter
configured in the L format. A wavelength/band pass of 495 nm/2 nm for excitation and
517 nm/1 nm for emission were utilized. All experiments were performed in 50 mM
HEPES, 100 mM NaCl at pH 7.5 in a Spectrosil far-UV quartz window fluorescence
cuvette (Starna Cells). Binding reactions were performed with 10 nM fluorescently
labeled DNA in the presence of 0.05 mg/mL bovine serum albumin (BSA) to prevent
68
adherence of DNA or protein to the cuvette walls. The anisotropy, r, was monitored
throughout the course of the binding assay in which protein was added in increments to
the fluorescently labeled DNA.
Each data point represents the average of 60 readings
taken over a period of 115 seconds. Anisotropy values were converted to fraction bound,
Fbound (fraction of DNA bound to peptide at a given DNA concentration), according to the
following equation:
Fbound 
r  r free
(rbound  r )Q  (r  r free )
where rfree is the anisotropy of fluorescently labeled DNA and rbound is the anisotropy of
the peptide-DNA complex at saturation. Q is the quantum yield that is applied as a
correction factor to account for changes in fluorescence intensity over the course of the
experiment (Q = Ibound/Ifree). Typically, Q values ranged from 0.9-1.0. Fbound was then
plotted against peptide concentration, and the data fit to a one-site binding model:
P+D
Kd 
Fbound 
PD
[ P][ D]
[ PD ]
Ptotal  Dtotal  K d  ( Ptotal  Dtotal  K d ) 2  4 Ptotal Dtotal
2 Dtotal
where P is the protein concentration and D is the DNA concentration.
3.2.8 Generation of Sequence Logos
A ‘Weblogo’ showing the conservation of the amino acid residues in all ZF domains (6
domains per protein) of R. norvegicus NZF-1, MyT1, and ST18 was generated using the
69
weblogo tool found at http://weblogo.berkeley.edu. The Weblogo of classical CCHH
ZFs was generated using http://prosite.expasy.org. A search of “zinc finger” was
performed and CCHH type ZFs was selected. The generated Weblogo was a result of
13,324 true positive hits from the UniProtKB/Swiss-Prot databank.(44-47)
3.3 Results and Discussion
3.3.1 Functional Roles of the Two Histidine Ligands within Each ZF of NZF-1
The published NMR structural data for NZF-1 and MyT1 are limited to single finger
domains of the proteins (F2 from NZF-1 and F5 from MyT1) (19, 27). In both of these
structures, the second conserved histidine within the ZF domain serves as the Zn(II)
coordinating ligand (19, 27). Remarkably, when this histidine is mutated to a noncoordinating residue (either alanine or glutamine) in either the second or third ZF domain
of NZF-1, the first histidine coordinates the Zn(II) in its place, suggesting that metal ion
coordination is flexible (19, 25). Here, we sought to understand the functional
consequences of histidine coordination in NZF-1. All of the structural and mutagenesis
studies that have been published are of single ZF domains, which do not bind to target
DNA with the requisite affinity (i.e. nM) to address this question (10). Thus, we prepared
a series of mutant proteins of a two domain construct of NZF-1, NZF-1-F2F3, for which
a high affinity DNA target sequence is known.
With this construct in hand, the
functional significance of each histidine residue was assessed.
70
(a)
APO
APO
2+ Co2+
Co
APO
APO
(b)
Zn2+ APO
Zn2+
APO
(c)
Zn2+
Co2+
Figure 3.2 Co(II) and Zn(II) titration of CCFHC (a) Plot of the change in the absorption
spectrum of 25 µM CCFHC between 500 and 800 nm as Co(II) is titrated. The titration was
performed in 200 mM HEPES, 100 mM NaCl buffer at pH 7.5. (b) Plot of the absorption
spectrum at 679 nm as either a function of added Co(II) to apo-CCFHC (blue) or Zn(II) to
Co(II)-CCFHC (red). The data were fit to appropriate binding equilibria and upper limit Kds of
5.2 ± 0.3 x 10-7 M and 3.2 ± 0.9 x 10-10 M for Co(II) and Zn(II), respectively were obtained.
The solid lines represent the non-linear least squares fit.
The first (H515 and H559) or second (H523 and H567) conserved histidine of
NZF-1-F2F3 were mutated to phenylalanines. The two mutant peptides were named
CCFHC and CCHFC, respectively. A third non-conserved histidine, H522, in F2 was
mutated to an alanine in order to prevent any metal ions binding adventitiously to this
residue in the mutant peptides (Figure 3.1b). The choice of phenylalanine was based upon
the proposal that the non-coordinating histidine is involved in a stacking interaction with
a conserved tyrosine residue (19). Phenylalanine in this position should preserve this
interaction.
71
3.3.2
Metal Binding Studies
To determine if the two mutant proteins, CCFHC and CCHFC, bound Zn(II), direct
titrations with Co(II) and competitive titrations with Zn(II) were performed. Co(II) is
routinely utilized as a spectroscopic probe for Zn(II) binding to ZFs (6, 48-53). Co(II),
which has a d7 electron count, exhibits distinct optical absorbances centered between
550-750 nm when it is coordinated to four ligands in a tetrahedral geometry (54). These
absorbance bands differ based upon the ligand set that coordinates Co(II), therefore the
nature of the ligands coordinating Co(II) in a given ZF can be deduced from these spectra
(55). Zn(II) typically binds more tightly to ZF domains than does Co(II), due to ligand
field stabilization energy (LFSE) differences.(14) Thus, Zn(II) binding to ZF sites can be
measured via a competitive titration of Co(II)-ZF with Zn(II) to reveal upper limit
dissociation constants (Kds) for each metal ion (14, 54, 56).
(a)
(b)
Zn2+
Co2+
Figure 3.3 Co(II) and Zn(II) titration of NZF-1-F2F3 (a) Plot of the change in the
absorption spectrum of NZF-1-F3F3 between 500 and 800 nm as NZF-1-F3F3 is titrated with
Co(II). The titration was performed in 200 mM HEPES/100 mM NaCl buffer at pH 7.5. (b)
Plot of the absorption spectrum at 679 nm as either a function of added Co(II) to apo-NZF-1F2F3 (blue) or Zn(II) to Co(II)-NZF-1-F2F3 (red). The data were fit to appropriate binding
equilibria and upper limit Kds of 7.0 ± 0.4 x 10-8 M and 1.2 ± 0.7 x 10-10 M for Co(II) and
Zn(II), respectively were obtained. The solid lines represent the non-linear least squares fit.
72
3.3.3 Co(II) Direct Titrations
Co(II) was titrated with NZF-1-F2F3, CCFHC and CCHFC. For all three proteins, d-d
bands between 550-750 nm were observed as Co(II) was added with maxima centered at
590, 650, and 679 nm. These d-d bands are from the 4A2 to 4T2(P) transition for a
tetrahedral geometry and the splitting into three components is due to lowered symmetry
(Figures 3.2, 3.3, and 3.4) (54, 57, 58). The shape of the spectra did not change during the
course of each titration, indicating that Co(II) binds to the two ZF sites with equal
affinities. The overall shape of the three split d-d bands for Co(II)-NZF-1-F2F3 and
Co(II)-CCFHC spectra were similar; while the d-d bands for the Co(II)-CCHFC were
split differently suggesting that the overall symmetry at the metal site is altered as a result
of the mutation (Figure 3.5a). The Co(II) titration data were fit to 1:1 binding equilibria,
and upper-limit Kds of 7.0 ± 0.4 x 10-8 M, 5.2 ± 0.3 x 10-7 M, and 3.7 ± 1.2 x 10-7 M were
(a)
(b)
Zn2+
Co2+
Figure 3.4 Co(II) and Zn(II) titration of CCHFC (a) Plot of the change in the absorption
spectrum of CCHFC between 500 and 800 nm as CCHFC is titrated with Co(II). The titration
was performed in 200 mM HEPES/100 mM NaCl buffer at pH 7.5. (b) Plot of the absorption
spectrum at 679 nm as either a function of added Co(II) to apo-CCHFC (blue) or Zn(II) to
Co(II)-CCHFC (red). The data were fit to appropriate binding equilibria and upper limit Kds
3.7 ± 1.2 x 10-7 M and 2.6 ± 1.2 x 10-9 M for Co(II) and Zn(II), respectively were obtained.
The solid lines represent the non-linear least squares fit.
73
determined for NZF-1-F2F3, CCFHC, and CCHFC, respectively. These affinities fit
within the range of reported binding affinities for ZF proteins (18, 25, 59).
3.3.4 Zn(II) Titrations
To determine the affinity of Zn(II) for NZF-1-F2F3, CCFHC and CCHFC, solutions of
each protein with 10-fold excess Co(II) was titrated with Zn(II). The decrease in the d-d
absorbances for the Co(II)-ZF spectra were monitored and the data were fit to a
competitive binding equilibrium. A representative fit for the CCFHC mutant is shown in
Figure 3.2. Upper limit Kds of 1.2 ± 0.7 x 10-10 M for NZF-1-F2F3, 3.2 ± 0.9 x 10-10 M
for CCFHC and 2.6 ± 1.2 x 10-9 M for CCHFC were determined (Figures 3.2b, 3.3b,
3.4b). These values fit well with those reported for other ZFs, including NZF-1 sites (18,
25, 59).
(a)
(b)
Figure 3.5 Comparison of NZF-1-F2F3 and Mutant Metal Binding and Fold (a) Overlay
of the absorbance spectra between 500-800 nm of Co(II)-NZF-1-F2F3, shown in purple,
Co(II)-CCFHC, shown in pink, and Co(II)- CCHFC, shown in blue. All experiments
performed in 200 mM HEPES, 100 mM NaCl, pH 7.5 (b) Overlay of the CD spectra of 20
µM Zn(II)-NZF-1-F2F3 shown in purple, 20 µM Zn(II)-CCFHC shown in pink, and 20 µM
Zn(II)- CCHFC shown in blue. All experiments performed in 25 mM Sodium Phosphate, pH
7.5.
74
3.3.5
CD Spectra of NZF-1-F2F3, CCFHC and CCHFC Mutants
To further characterize the effect of mutating either the coordinating or noncoordinating histidine on the structure of NZF-1-F2F3, CD spectra of the three proteins
were measured (Figures 3.5b and 3.6). In the absence of metal ions, all three proteins
have similar spectra that are characterized by a negative signal at 200 nm, typical of a
random coil (53, 60-62). In the presence of either stoichiometric Co(II) or Zn(II), the only
additional feature is a negative signal centered at 220 nM. These features were expected
(a)
(b)
(c)
Figure 3.6 Comparison of apo-, Co(II)-, and Zn(II)-Bound NZF-1-F2F3 Peptides (a)
Overlay of the CD spectra of 20 µM apo-NZF-1-F2F3 represented by the solid line, 20 µM
Co-(II)-NZF-1-F2F3 represented by the dashed line, 20 µM Zn-(II)-NZF-1-F2F3
represented by the dotted line. (b) Overlay of the CD spectra of 20 µM apo-CCFHC
represented by the solid line, 20 µM Co-(II)-CCFHC represented by the dashed line, 20 µM
Zn-(II)-CCFHC represented by the dotted line. (c) Overlay of the CD spectra of 20 µM
apo-CCHFC represented by the solid line, 20 µM Co-(II)-CCHFC represented by the
dashed line, 20 µM Zn-(II)-CCHFC represented by the dotted line. All experiments
performed in 25 mM Sodium Phosphate, pH 7.5.
75
as the NMR structures of NZF-1 and MyT1 (19, 27) do not exhibit significant alpha
helical or beta sheet content and they match spectra recently reported for a single ZF of
MyT1 by Wilcox and co-workers (53). Notably, the CD spectra of the Co(II) or Zn(II)
bound CCHFC mutant are slightly different than the analogous NZF-1-F2F3 and CCFHC
spectra, as shown in figure 3.5b and 3.6. This offers further evidence that the CCHFC
mutant adopts a different fold upon metal coordination than NZF-1-F2F3 and CCFHC.
3.3.6 DNA Binding Studies to Define the Functional Role(s) of the Histidine Ligands
To understand the roles of the two conserved histidine residues on function, fluorescence
anisotropy (FA) was utilized to measure the affinity of NZF-1-F2F3 and the two mutants,
CCFHC and CCHFC for the β-retinoic acid receptor (β-RAR) recognition sequence (28,
52, 63-65). The native protein, NZF-1-F2F3 was expected to bind to this DNA with
nanomolar affinity, based upon previous studies (28). Similarly, the CCFHC mutant,
which maintains the metal coordinating histidine, was also expected to bind to this DNA
Figure 3.7 Fluorescence Anisotropy of NZF-1-F2F3 Variants. Comparison of the
change in anisotropy (as fraction bound) as NZF-1-F2F3, shown in closed purple
circles, CCFHC, shown in closed pink circles, and CCHFC, shown in closed blue
diamonds are titrated in 10 nM fluorescently labeled β-RAR DNA. An example of a
control titration (NZF-1-F2F3 with a randomized segment of DNA) is shown in open
purple circles. All experiments were performed in triplicate in 50 mM HEPES, 100 mM
NaCl pH 7.5.
76
target with nM affinity. In contrast, the CCHFC mutant was expected to show different
DNA binding properties, because the coordinating histidine has been abrogated. NZF-1F2F3 selectively bound to β-RAR with a Kd of 1.4 ± 0.2 x10-8 M, as expected;
surprisingly, DNA binding was not observed for either mutant (Figure 3.7). None of the
proteins bound to a random DNA sequence.
The abrogation of DNA binding for CCHFC was expected, as in this mutant
Zn(II) is coordinated to a different histidine and the overall protein fold has been
perturbed. However, the lack of DNA binding for the CCFHC mutant was unexpected,
because this mutant was designed to retain the key elements thought to be important for
the protein’s fold. These included the native coordinating histidine and a phenylalanine at
the position of the non-coordinating histidine. From structural studies, this noncoordinating histidine had been proposed to be part of a stacking interaction with a noncoordinating tyrosine, which helps stabilize the structure (Figure 3.1c, Y520) (19). The
Non metalcoordinating
histidine
Tyrosine
Figure 3.8 Model of mMyT1 interacting with the βRAR DNA, shown in purple
(PDBID 2JX1). Metal coordinating residues shown in blue; residues shown to stack in
NZF-1 structure shown in red; residue not conserved between NZF-1-F2F3 and MyT1F2F3 shown in green. Figure generated in Pymol.
77
DNA binding studies reported here reveal that the role of this histidine is more complex
than just participation in a stacking interaction. The non-coordinating histidine appears to
be critical for DNA binding, and we hypothesize two potential roles for this histidine: (i)
it may play a direct role in DNA recognition or (ii) it may play an indirect role in DNA
binding by stabilizing a residue, such as the tyrosine it has been proposed to ‘stack’ with,
via additional bonding interactions that may have been disrupted via the mutation of the
histidine residue to phenylalanine (e.g. hydrogen bonding). Although there are no
structures of NZF-1 bound to DNA, Gamsjaeger and co-workerrs have modeled a
structure for a single ZF domain of MyT1 bound to DNA with the Haddock docking
program (Figure 3.8). In this model, both the non-coordinating histidine and the tyrosine
are positioned to interact with the DNA (27). Furthermore, in some of the ZFs domains in
this family, arginine is found in the place of tyrosine. Arginine often serves as a DNA
(a)
(b)
Zn2+
Co2+
Figure 3.9 Co(II) and Zn(II) titration of MyT1-F2F3 (a) Plot of the change in the
absorption spectrum of MyT1-F2F3 between 500 and 800 nm as My T1-F2F3 is titrated with
Co(II). The titration was performed in 200 mM HEPES/100 mM NaCl buffer at pH 7.5. (b)
Plot of the absorption spectrum at 679 nm as either a function of added Co(II) to apo-MyT1F2F3 (blue) or Zn(II) to Co(II)-MyT1-F2F3 (red). The data were fit to appropriate binding
equilibria and upper limit Kds 2.0 ± 1.1 x 10-7 M and 3.6 ± 2.0 x 10-10 M for Co(II) and
Zn(II), respectively were obtained. The solid lines represent the non-linear least squares fit.
78
recognition element in DNA binding proteins (66-68).
3.3.7 Switching the DNA Binding Properties of MyT1 to those of NZF-1
MyT1 shows a high level of sequence similarity to NZF-1, although MyT1 is found in
different cells and regulates different genes than NZF-1 (21, 29, 30). The DNA target
sequence recognized by MyT1 has not yet been identified, but has been proposed to be
the same target sequence as NZF-1 (24). The singular report of solution-based DNA
binding studies for MyT1 utilized NZF-1’s β-RAR target sequence, and the reported
affinities of MyT1 for this DNA sequence were weaker than the affinity of NZF-1-F2F3
for this same DNA sequence (27). It has been proposed that the motif recognized by
NZF-1 within the β-RAR sequence is an AAGTT sequence (18, 30). This sequence is not
present in all of the promoter sequences of the genes recognized by MyT1, (42) thus
(a)
(b)
Zn2+
Co2+
Figure 3.10 Co(II) and Zn(II) titration of Q291R MyT1-F2F3 (a) Plot of the change in
the absorption spectrum of Q291R MyT1-F2F3 between 500 and 800 nm as Q291R MyT1F2F3 is titrated with Co(II). The titration was performed in 200 mM HEPES/100 mM NaCl
buffer at pH 7.5. (b) Plot of the absorption spectrum at 679 nm as either a function of added
Co(II) to apo-Q291R MyT1-F2F3 (blue) or Zn(II) to Co(II)-Q291R MyT1-F2F3 (red). The
data were fit to appropriate binding equilibria and upper limit Kds of 9.3 ± 1.3 x 10 -8 M and
2.1 ± 0.8 x 10-9 M for Co(II) and Zn(II), respectively were obtained. The solid lines
represent the non-linear least squares fit.
79
Figure 3.11 Comparison of Fold of MyT1-F2F3 Variants and NZF-1-F2F3. Overlay of
the CD spectra of 20 µM Zn(II)-NZF-1-F2F3 shown in purple, 20 µM Zn(II)-MyT1-F2F3
shown in red, and 20 µM Zn(II)-Q291R MyT1-F2F3 shown in green. All experiments
performed in 20 mM Sodium Phosphate, pH 7.5.
MyT1 likely recognizes a different DNA target than NZF-1.
Given the high sequence identity between NZF-1 and MyT1, we hypothesized that
the residues that are not conserved between NZF-1 and MyT1 must determine DNA
sequence selectivity. By aligning the NZF-1-F2F3 sequence with the analogous region of
MyT1 (MyT1-F2F3) we found that within the ZF domains only two amino acids differ
between the two sequences (Figure 3.1b). One of these amino acids is a serine residue in
NZF-1-F2F3 and a threonine residue in MyT1-F2F3. As these amino acids have very
similar properties, we hypothesized these residues would play similar functional roles. In
contrast, an arginine in NZF-1-F2F3 is a glutamine in MyT1-F2F3. Arginine residues are
often implicated in DNA recognition and therefore this amino acid may be the key switch
for the high affinity βRAR DNA recognition by NZF-1 (66-68). As such, a mutation of
the glutamine in MyT1-F2F3 to arginine is predicted to result in high affinity DNA
binding. To test this hypothesis, both native MyT1-F2F3 and Q291R MyT1-F2F3 were
prepared and characterized.
80
3.3.8 Metal Binding and Folding Studies
Direct Co(II) titrations and competitive Zn(II) titrations of MyT1-F2F3 and Q291R
MyT1-F2F3 were performed analogously to the studies described above for NZF-1-F2F3
and its mutants. The Co(II) spectra for both proteins matched those observed for NZF-1F2F3 and CCFHC, indicating that the same ligands are involved in metal ion
coordination for both MyT1 and NZF-1 (Figures 3.9 and 3.10). The Co(II) data were fit
to 1: 1 binding equilibria and upper-limit Kds of 2.0 ± 1.1 x 10-7 M and 9.3 ± 1.3 x 10-8 M
were determined for MyT1-F2F3 and Q291R MyT1-F2F3 respectively, The Zn(II) data
were fit to a competitive equilibria and upper-limit Kds of 3.6 ± 2.0 x 10-10 M and 2.1 ±
0.8 x 10-9 M were determined. The CD spectra were also similar to that reported for NZF1-F2F3 (Figure 3.11 and 3.12).
(a)
(b)
Figure 3.12 Comparison of apo-, Co(II)-, and Zn(II)-Bound MyT1-F2F3 Peptides (a)
Overlay of the CD spectra of 20 µM apo-(II)-MyT1-F2F3 represented by the solid line, 20
µM Co-(II)- MyT1-F2F3 represented by the dashed line, 20 µM Zn-(II)- MyT1-F2F3
represented by the dotted line. (b) Overlay of the CD spectra of 20µM apo-(II)-Q291R
MyT1-F2F3 represented by the solid line, 20 µM Co-(II)- Q291R MyT1-F2F3 represented
by the dashed line, 20 µM Zn-(II)- Q291R MyT1-F2F3, represented by the dotted line. All
experiments performed in 25 mM Sodium Phosphate, pH 7.5.
81
3.3.9 DNA Binding Studies of MyT1-F2F3 and Q291R MyT1-F2F3 with β-RAR
We first performed FA studies of MyT1-F2F3 with β-RAR, to quantify the binding
affinity (Figure 3.13). The only previous study of MyT1-F2F3 with this DNA target
utilized surface plasmon resonance (SPR) and only a semi-quantitative affinity was
reported (27). As predicted, MyT1-F2F3 bound to β-RAR with a significantly weaker
affinity, Kd of 1.3 ± 0.11 x10-6 M, than the affinity of NZF-1-F2F3 for this same target
(Kd of 1.4 ± 0.2 x10-8 M). Notably, the affinity of MyT1-F2F3 for a random sequence of
DNA that was used as a control was on the same order of magnitude (Kd = 9.1 ± 0.5 x
10-7 M) as its measured affinity for β-RAR. This indicates that the binding observed for
MyT1-F2F3 with β-RAR is non-specific. In contrast, when the affinity of the mutated
MyT1 protein Q291R for β-RAR was measured, a Kd of 3.3 ± 0.12 x 10-8 M was
determined. Remarkably, this affinity is on the same order of magnitude as the affinity of
Figure 3.13 Fluorescence Anisotropy of MyT1-F2F3 Variants. Change in anisotropy
(as fraction bound) as NZF-1-F2F3, shown in closed purple circles, and Q291R MyT1F2F3, shown in closed green circles, are titrated into β-RAR DNA. Example of the
control as Q291R MyT1-F2F3 is titrated into a random segment of DNA is shown in
open green circles. Inset shows change in anisotropy as MyT1-F2F3 is titrated in β-RAR
(closed red circles) and random DNA segment (open red circles). 10 nM fluorescently
labeled DNA used for all experiments. All experiments were performed in triplicate in 50
mM HEPES, 100 mM NaCl pH 7.5.
82
NZF-1-F2F3 for this DNA target. Thus, mutation of a single glutamine in MyT1-F2F3 to
arginine results in high affinity, specific DNA binding to β-RAR which is on par with the
native NZF-1-F2F3’s binding affinity.
3.4 Conclusions
The results of the studies reported herein provide key insight into two overarching
questions regarding NZF-1 and MyT1 function: (1) what is the functional role of the noncoordinating histidine that is present in all CCHHC type ZF proteins? and (2) How do
NZF-1 and MyT1 discriminate between DNA targets, given the high sequence identity
between individual ZFs? By mutating the coordinating and non-coordinating histidine
residues in a functional form of NZF-1 (NZF-1-F2F3), we discovered that both histidines
must be present in order for the protein to recognize target DNA. Thus, the noncoordinating histidine plays a critical role in DNA recognition - possibly via a stacking
interaction with DNA bases or via the stabilization of other essential DNA interacting
residues. In addition, we have discovered that MyT1-F2F3 can be programmed to bind to
the NZF-1-F2F3 DNA target with high affinity and specificity via a single point
mutation.
NZF-1 and MyT1 each contain multiple, highly similar ZF domains that are
organized in clusters (Figure 3.1a) (29, 30). The DNA recognition sequence has only
been defined for the F2F3 cluster of NZF-1 (18). The role(s) of the additional ZF clusters
found in NZF-1 in DNA recognition are not known and bona fide DNA target sequences
for MyT1 have not yet been identified. Given the high degree of sequence similarity
between the ZFs, it is tempting to propose that the ZF domains all recognize the same
83
DNA sequence. However, as we report here, MyT1-F2F3 does not bind to NZF-1-F2F3’s
DNA target sequences with high affinity. Similarly, MacKay and co-workers have
reported weaker binding of MyT1-F4F5 for NZF-1-F2F3’s DNA target (27). Our
discovery that a single point mutation at one of the few less-conserved residues renders
MyT1-F2F3 capable of binding to NZF-1’s target DNA suggests that the ZF clusters each
recognize different DNA target sequences and it is the handful of less-conserved residues
within the amino acid sequence of the ZF domains that determine which DNA sequence
is recognized. (Figure 3.14a). This proposed model of DNA target recognition by NZF1 and MyT1 contrasts the DNA binding paradigm that is operative for classical Cys2His2
ZFs. For classical ZFs, only a few amino acids are conserved between the sequences of
homologs and these conserved amino acids are essential for the fold of the protein (69).
There is great variability in the sequence of classical ZFs outside of these residues and
this variability allows for alternate DNA recognition properties (Figure 3.14b). Four key
(a)
(b)
Figure 3.14 Comparison of Conserved Amino Acids in CCHHC and CCHH
Domains (a) Weblogo generated from alignment of all CCHHC domains from NZF-1,
MyT1, and ST18. Height of amino acid residue corresponds to degree of conservation
(b) ExPASy output of all known CCHH ZF domains.
84
positions in these ZF domains have been implicated in being essential in DNA
recognition, while single mutations in the ZF domains of the NZF-1 proteins appear to
completely alter DNA recognition properties (10). The CCHHC family of non-classical
ZF proteins are emerging as a new type of ZF protein in which ZF domains contain
highly conserved residues that are likely important for protein fold but do not appear to
drive DNA recognition. Rather, it is the few less-conserved residues that appear to dictate
this DNA recognition. As such, these CCHHC ZFs appear to utilize a completely
different mode of DNA recognition than the classical ZF proteins. Further work to
understand the determinants of DNA recognition for the other ZF domains of both NZF-1
and MyT1 are in progress.
85
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95
Chapter 4
A role for hydrogen bonding in DNA recognition by the non-classical
CCHHC type zinc finger, NZF-1*
4.1 Introduction
Zinc finger (ZF) proteins are zinc co-factored metalloproteins that are critical for a
myriad of biological processes, most notably transcriptional regulation (1, 2). There are at
least fourteen distinct classes of ZFs (3). All ZFs contain one or more discrete ZF
domains that include cysteines and/or histidines, which serve as ligands for zinc
coordination (1, 4, 5). One ZF family, collectively known as the ‘CCHHC’ type ZF
family, is unique in that it contains five potential zinc ligands: Cys2His2Cys within each
ZF domain (1). Three members of this family have been identified to date: Neural Zinc
Finger Factor-1 (NZF-1), Myelin Transcription Factor 1 (MyT1) and Suppression of
Tumorgenicity 18 (ST18) (6-8). NZF-1 and MyT1 are essential for the development of
the central nervous system where they function to control the development of neurons
and oligodendrocytes via regulation of the β-retinoic acid receptor (βRAR) and the
proteolipid protein, respectively (6, 7).
ST18 is associated with controlling tumour
development, although its specific role in this process has not yet been established (9).
*Adapted from Besold, A.N.; Amick, D.L.; Michel, S.L.J. 2014. Mol. Biosyst.,
10(7):1753-6.
96
(a)
(b)
(c)
(d)
(e)
Figure 4.1 Zinc Finger Domains of the NZF Family (a) Cartoon depicting the arrangement
of the ZF domains of NZF-1 with NZF-1-F2F3 boxed. (b) Alignment of ZF domains of NZF1-F2 and NZF-1-F3 with conserved CCHHC residues shown in purple and non-conserved
residues denoted in black. (c) NMR structure of NZF-1-F2, with metal coordinating ligands in
cyan (PDBID: 1PXE). (d) A zoomed in view of the local structure surrounding the noncoordinating histidines found in NZF-1-F2 (PDBID: 1PXE) and MyT1-F5 (PDBID: 2JYD).
(e) Zoomed in view of the predicted structure of NZF-1-F3 centred at the non-coordinating
histidine. All structures were generated using PyMOL.
All CCHHC family ZFs contain multiple, highly homologous, CCHHC domains
arranged in clusters (Figure 4.1a) (1). A bona fide DNA target has only been identified
for the cluster of NZF-1 made up of ZF domains #2 and #3 (named NZF-1-F2F3) (6, 10).
We have shown that the few non-conserved amino acid residues present in NZF-1-F2F3
are critical for sequence specific DNA binding (11). This model of DNA recognition
contrasts the paradigm for classical ZFs: classical ZFs utilize a Cys2His2 ligand set for
97
Zn(II) coordination and there are only a few additional conserved amino acids that are
critical for the fold and function of the protein (1, 11-13).
Although the CCHHC ZFs have five potential Zn(II) coordinating ligands per
domain (3 Cys, 2 His; Figure 4.1b), studies have shown that only four ligands directly
coordinate Zn(II) (Figure 4.1c). These ligands are the three cysteines and the second
conserved histidine resulting in a CCHC ligand set (14-16). The role of the first
conserved (non-Zn(II)-coordinating) histidine is not understood. The NMR structure of
just finger 2 of NZF-1 (NZF-1-F2) has been reported and the non-coordinating histidine
appears to be involved in a pi-stacking interaction with a tyrosine residue to stabilize the
fold around the zinc ion such that the protein can bind to DNA (Figure 4d) (15).
However, we found that when this histidine is mutated to a phenyalanine in a functional
construct of NZF-1 (NZF-1-F2F3), DNA binding was completely abolished, indicating
that the role of the histidine is not a simple pi-stacking interaction (11). Moreover, in the
structure of F5 of MyT1, which has 80% sequence homology to F2 of NZF-1, the
histidine and tyrosine are not oriented in a position that allows for pi-stacking (Figure
4.1d) (14, 17). Both structures, NZF-1-F2 and MyT1-F5, lack significant secondary
structure and principally fold into a series of flexible loops. Circular dichroism studies on
different ZFs of this family confirm these results (11, 18). Thus, the differences in
orientations between the histidine and tyrosine residues observed in the two NMR
structures indicate that the overall folds of these domains are highly flexible.
Careful inspection of the sequences of F2 and F3 of NZF-1 revealed that the
tyrosine residue that sometimes appears to pi-stack with the non-coordinating histidine is
absent in F3. An arginine is found in place of this residue. To better understand the
98
interactions taking place in F3, a model of NZF-1-F3 was generated in PyMOL with the
NZF-1-F2 structure as a scaffold (Figure 4.1e) (19). Arginine residues often participate in
hydrogen bonding interactions, but are not involved in pi stacking interactions.(20) Thus,
the non-coordinating histidine may participate in hydrogen bonding, rather than pistacking, as histidine can form hydrogen bonds with either tyrosine or arginine but can
only form pi stacking interactions with tyrosine.
Mutants of NZF-1-F2F3 were prepared to assess the role of hydrogen bonding in
function. The non-coordinating histidines from F2 (H515) and F3 (H559) of NZF-1 were
mutated to glutamine producing three mutants: NZF-1-F2F3-H515Q, NZF-1-F2F3H559Q, and NZF-1-F2F3-H515/559Q (called H515Q, H559Q and H515/559Q). The
mutations are designed to retain hydrogen-bonding interactions, but disrupt pi-stacking.
4.2 Materials and Methods
4.2.1 Design of NZF-1-F2F3 Mutant Peptides
A single point mutation of histidine 515 and/or histidine 559 to glutamine was
accomplished using a QuikChange Mutagenesis Kit (Agilent). The wild type (WT) NZF1-F3F3 DNA corresponding to amino acids 487 – 584 of the full length protein from
Rattus norvegicus ligated into a pET15b vector in which the hexahistidine tag had been
removed, using the restriction sites NcoI and BamHI, served as the template for the
mutagenesis. The mutant sequences were confirmed via DNA sequencing performed at
the Biopolymer/Genomic Core Facility housed at the University of Maryland School of
Medicine.
99
4.2.2 Expression and Purification of NZF-1-F2F3 Mutant Peptides
All peptides were expressed and purified in a similar manner to WT NZF-1-F2F3.
Briefly, the pET15b vector containing the gene of interest was transformed into
BL21(DE3) Escherichia coli (Novagen) cells. Overnight cultures were grown in Luria –
Bertani (LB) broth containing 100 µg/mL ampicillin at 37°C. These cells were used to
inoculate a 1L culture of LB broth containing 100 µg/mL ampicillin. Cells were grown to
mid-log phase at 37°C and expression was induced using 1 mM isopropyl β-D-1thiogalactopyranoside (IPTG; Research Products International Corp). Cells were grown 4
hours post-induction and were harvested by centrifugation for 15 minutes at 7800g and
4°C. Cell pellets were resuspended in 25 mM Tris [tris(hydroxymethyl)-aminomethane]
at pH 8.0, 100 μM ZnCl2, and 5 mM dithiothreitol (DTT) containing a mini
ethylenediaminetetraacetic acid (EDTA) free protease inhibitor tablet (Roche). Cells were
then lysed via sonication on a Sonic Dismembrator Model 100 (Fisher) and cell debris
was removed by centrifugation at 12,100g for 15 minutes at 4°C. The peptide of interest
was purified from the bacterial supernatant using a stepwise salt gradient from 25 mM –
1 M sodium chloride on a SP Sepharaose Fast Flow column (Sigma). The cysteine thiols
of the peptide were reduced by incubation with 10 mM tris(2-carboxyethyl)phosphine
(TCEP) (Thermo) for 30 minutes at room temperature. The solution was then filtered and
further purified using High Performance Liquid Chromatograph (HPLC) on a Waters 626
LC system and a Waters Symmetry Prep 300 C18 7 μm reverse phase column. An
acetonitrile gradient was used containing 0.1% trifluoroacetic acid (TFA). The peptides
eluted at 29% acetonitrile with 0.1% TFA. The purified peptides were lyopholized using
a Thermo Savant SpeedVac concentrator housed in a Coy anaerobic chamber (97%
100
(b)
(a)
15 kDa
6 kDa
10.7
kDa
(d)
(c)
(d)
Figure 4.2 HPLC Purified Peptides. Pure peptide used for studies boxed in blue. (a)
WT NZF-1-F2F3 (b) H515Q (c) H559Q (d) H515/559Q (e) NZF-1-F2.
N2/3% H2). Although ZF peptides tend to smear when run on SDS-PAGE gels, purity
analysis can be done using this method. Only peptides judged to be >95% pure via SDS
PAGE were used in studies (Figure 4.2). The yield of WT, H515Q, H559Q and
H515/559Q of NZF-1-F2F3 as well as a single finger peptide, NZF-1-F2, were 2.6
mg/mL, 5.6 mg/mL, 4.7 mg/mL, 5.1 mg/mL, and 21 mg/ml, respectively.
4.2.3 Metal Binding Studies
Metal ion titrations were performed using a PerkinElmer Lambda 25 UV−visible
Spectrometer. 20 – 25 µM apo-peptide was titrated with 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4,
1.6, 1.8, 1.9, 2.0, 5.0, 10.0, 15.0, and 20.0 equivalents of CoCl2 (Fisher Scientific) and the
101
appearance of the d-d transitions was monitored. ZnCl2 (Fisher Scientific) was then
titrated into the Co(II)-peptide and the disappearance of the d-d transitions was monitored
as Zn(II) displaced Co(II) in the peptide. All titrations were performed in 200 mM
HEPES, 100 mM NaCl, pH 7.5. A plot of A679 versus metal concentration was fit to a 1:1
binding model using non-linear least squares analysis (KaleidaGraph, Synergy Software)
and an upper limit dissociation constant (Kd) for the metals were determined.
P + Co
Kd =
PCo
[P][Co]
[PCo]
[P]total +[Co]total + K d - ([P]total +[Co]total + K d )2 - 4[P]total [Co]total
[PCo] =
2[Co]total
4.2.4 Electron Paramagnetic Resonance (EPR) Spectroscopy
Electron paramagnetic resonance (EPR) spectra were collected on a Bruker EMX EPR
Spectrometer controlled with a Bruker ER 041 XG microwave bridge at 12 K.
Temperature was maintained with a continuous-flow liquid He cryostat and an ITC503
temperature controller (Oxford Instruments, Inc). The spectrometer parameters were
microwave frequency, 9.47 GHz; microwave power, 2 mW; magnetic field amplitude, 10
G. Samples were prepared anaerobically in a Coy inert atmospheric chamber (97% N2;
3% H2). 400 – 500 μM Co(II)-NZF-1-F2F3 WT and mutant peptides were prepared in 50
mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 100 mM NaCl, pH
7.5. The final sample was mixed with glycerol (1:1, by volume).
102
Samples were
transferred to a Quartz 4 mm OD EPR cell with a rubber septum and the samples were
frozen in liquid nitrogen for storage prior to the experiment.
4.2.5 Oligonucleotide Probes
Oligonucleotides were purchased from Integrated DNA Technologies, Inc. (Coralville,
IA), in the HPLC purified formed with a 5’ end labeled fluorescein. The end labeled
strand of the β-retinoic acid receptor (βRAR), CACCGAAAGTTCACTC, was purchased
along
with
its
anti-sense
unlabeled
complement.
A
random
DNA
strand,
TGTTTCTGCCTCTGT, was purchased in a similar matter. The oligonucleotides were
annealed by mixing a ratio of 1.25:1 unlabeled:labeled in 10 mM Tris, 10 mM NaCl, pH
8.0. The solution is placed in a water bath set to 10°C higher than the melting
temperature of the DNA strands. The annealing reaction was allowed to proceed for 5
minutes prior to the water bath being turned off and subsequently allowed to cool
overnight. The resultant double stranded DNA were then quantified and stored at -20°C
until use.
4.2.6 Fluorescence Anisotropy (FA)
Fluorescence Anisotropy assays were performed on an ISS PC-1 spectrofluorometer
configured in the L format. A wavelength/band pass of 495 nm /2 nm for excitation and
517 nm /1 nm for emission were used. Titrations were performed in 50 mM HEPES, 100
mM NaCl, pH 7.5 in the presence of 0.05 mg/mL of bovine serum albumin (BSA) to
prevent adherence of the DNA or peptide to the cuvette walls. All experiments were
performed using Spectrosil far-UV quartz window fluorescence cuvettes (Starna Cells).
103
The anisotropy, r, was monitored as peptide was titrated into 10 nM fluorescently labeled
DNA. Each data point represents the average of 60 readings taken over the period of 115
seconds. The change in anisotropy was plotted versus peptide concentration and the data
was fit to a one-site binding model:
P+D
Kd 
PD 
PD
[ P][ D]
[ PD ]
Ptotal  Dtotal  K d  ( Ptotal  Dtotal  K d ) 2  4 Ptotal Dtotal
2 Dtotal
4.2.7 Generating PyMOL Models
Models of NZF-1-F3, H515Q, and H559Q were generated in PyMOL using the NZF-1F2 solution structure (PDBID: 1PXE) as a scaffold. The peptide was displayed as a
“cartoon” and the metal coordinating side chains along with H515 and Y520 were
displayed as “sticks.” To create NZF-1-F3, the “mutagenesis” tool was employed and
Y520 was chosen as the amino acid to mutate. The most likely backbone dependent
rotamer of arginine was chose to replace the tyrosine in order to mimic the wild type
NZF-1-F3. H515Q was created in a similar manner, using the mutagenesis tool to
mutated H515 into the most likely backbone dependent rotamer of glutamine. The NZF1-F3 model was used as the scaffold to create the H559Q mutant in which the non-metal
coordinating histidine was mutated to the most likely backbone dependent rotamer of
glutamine. The distance between two non-hydrogen atoms were measured using the
“measurement” tool.
104
4.3 Results and Discussion
To determine whether the mutants bound Zn(II), optical titrations using Co(II) as a
spectroscopic probe for Zn(II) were performed. This is a common strategy used to
investigate metal ion coordination to ZF sites (21-23). Co(II), like Zn(II), can coordinate
cysteine and histidine ligands in a tetrahedral geometry but has the advantage of having a
partially filled d-shell (d7) which allows for optically observable d-d transitions. The
optical spectrum for a Cys2HisCys site is expected to exhibit transitions between 550 –
750 nm (1, 24). All three mutant peptides were titrated with Co(II) and the expected d-d
bands were observed (Figure 4.3; Figure 4.4). The intensity of the d-d transitions became
saturated after 2 equivalents of Co(II) were added, indicating the expected 2:1
Co(II):peptide binding stoichiometry. No intermediate spectra were observed during the
course of the titrations, which suggests that both ZFs within each domain bind to Co(II)
with equal affinity. The data were fit to a 1:1 binding equilibrium, treating the two sites
(a)
(b)
(c)
Figure 4.3 Co(II) and Zn(II) titration of H515Q (a) Cartoon of ZF titrations. (b) Plot of the
change in the absorption spectrum between 500-800 nm as Co(II) and Zn(II) are titrated with
25 µM H515Q. (c) Plot of the absorbance at 679 nm as a function of added Co(II) (blue) or
Zn(II) (red). The solid line represents non-linear least squares fits to 1:1 and competitive
binding equilibria, respectively.
105
(a)
(c)
(b)
(d)
Figure 4.4 Co(II) and Zn(II) titration of H559Q and H515/559Q (a) Plot of the
change in the absorption spectrum between 500-800 nm as Co(II) and Zn(II) are titrated
with 25 µM H559Q. (b) Plot of the absorbance at 679 nm as a function of added Co(II)
(blue) or Zn(II) (red) to H559Q. The solid line represents non-linear least squares fits to
1:1 and competitive binding equilibria, respectively. (c) Plot of the change in the
absorption spectrum between 500-800 nm as Co(II) and Zn(II) are titrated with 21 µM
H515/559Q. (d) Plot of the absorbance at 679 nm as a function of added Co(II) (blue) or
Zn(II) (red) to H515/559Q. The solid line represents non-linear least squares fits to 1:1
and competitive binding equilibria, respectively.
equally (23). Upper limit dissociation constants (Kds) of 6.9 ± 0.3 x 10-7, 5.2 ± 1.2 x 10-7,
and 3.9 ± 1.0 x 10-7 M were determined for Co(II) binding to H515Q, H559Q, and
H515/559Q, respectively. To determine the affinities of the peptides for Zn(II), solutions
of the peptides with 20 excess molar equivalents of Co(II) were titrated with Zn(II) and
the decrease in the d-d absorbance bands monitored. These data were fit a competitive
binding model (23, 24) and upper limit Kds for Zn of 3.0 ± 0.9 x 10-9 , 5.1 ± 2.1 x 10-10 ,
and 1.2 ± 0.6 x 10-11 M were determined for H515Q, H559Q, and H515/559Q,
106
Table 4.1 Upper limit dissociation constants for Co(II) and Zn(II) binding to NZF-1-F2F3
WT and mutant peptides along with their dissociation constants for binding to βRAR.
Peptide
Kd Co(II) (M)
Kd Zn (II) (M)
Kd to βRARE (M)
WT
7.0 ± 0.4 x 10-8 1.2 ± 0.7 x 10-10
1.4 ± 0.2 x 10-8
H515Q
H559Q
6.9 ± 0.3 x 10-7 3.0 ± 0.9 x 10-9
5.2 ± 1.2 x 10-7 5.1 ± 2.1 x 10-10
3.0 ± 0.2 x 10-6
3.7 ± 0.3 x 10-6
H515/559Q
3.9 ± 1.0 x 10-7 1.2 ± 0.6 x 10-11
n.b.
respectively. These Kds are in the same range as those reported for other wild type (WT)
and mutant NZF-1 constructs indicating that the mutations have not affected the peptides’
ability to bind metal ions (Table 4.1) (25).
The Co(II)-peptides were further characterized by X-band Electron paramagnetic
resonance (EPR) spectroscopy at 12 K. Co(II)-WT exhibited a broad spectrum indicative
of high spin Co(II). Rhombic symmetry was observed with g values of 5.36, 2.96, and
2.31 (Figure 4.5a). No observable hyperfine splitting was noted. The spectrum is
consistent with spectra reported for other tetrahedral Co(II) coordination complexes (26-
(a)
(b)
Figure 4.5 Metal Center and DNA Binding Analysis of NZF-1-F2F3 Variants (a)
EPR spectrum of Co(II)-NZF-1-F2F3, table of g values for WT and variants (inset). (b)
Comparison of anisotropy as H515Q (blue), H559Q (green), and H515/559Q (red) are
titrated with βRAR.
107
(a)
(b)
Figure 4.6 EPR Analysis of Double Mutants (a) EPR spectra of Co(II)-H515/559Q. (b)
EPR spectra of Co(II)-H523/567F.
29). The EPR spectrum of H515/559Q was identical to that of the WT peptide (Figure
4.6a), suggesting that the mutations did not affect the geometry at the metal centers. The
EPR spectrum of a previously reported mutant in which the metal coordinating histidine
had been mutated such that the non-coordinating histidine now bound Co(II) (named
NZF-1-F2F3-H523/567F) (11) was also recorded to determine how switching the
coordinating ligands affects the metal centre. The spectrum showed only slight
differences, indicating that NZF-1 forms high-spin Co(II) species when four coordinating
ligands are present (Figure 4.6b).
Fluorescence anisotropy (FA) of the mutants with RAR DNA, which is NZF-1’s
physiological target, and a random segment of DNA was performed to measure the
effects of the mutations on DNA binding (11, 24). H515Q and H559Q bound the βRAR
DNA specifically with Kds of 3.0 ± 0.2 x 10-6 M and 3.7 ± 0.3 x 10-6 M, respectively;
whereas, the double mutant, H515/559Q, did not bind to DNA with measurable affinity
(Table 4.1, Figure 4.5b). In comparison, WT binds βRAR DNA with an affinity of 1.4 ±
0.2 x 10-8 M (11)
108
There are several possible interpretations of these results. One explanation is that
for the single ZF domain mutants, only the non-mutated domain is binding DNA and by
inference when both domains are mutated no binding would be observed. There are some
examples of single ZF domains that can bind to DNA or RNA targets (30-32). These
single ZFs typically exhibit Kds in the micromolar regime (32). To investigate this, a
single ZF domain of NZF-1, NZF-1-F2, was prepared using published protocols (15) and
DNA binding was measured. NZF-1-F2 did not bind to either the βRAR or the random
DNA (data not shown). Thus, the explanation that mutation of a single ZF domain merely
abrogates binding from that mutated domain was ruled out.
A second explanation is that hydrogen bonding is but one of several properties
required for proper fold and function of NZF-1-F2F3. To further understand the structural
(a)
(b)
(c)
(d)
Figure 4.7 Hydrogen Bond Analysis Close up of (a) F2: H515 and Y520; (b) F3: H559
and R564; (c) H515Q and Y520; (d) H559Q and R564. Distances of potential H bond
shown adjacent to bond (shown in green).
109
consequences of the H to Q mutants, the structures of the mutant ZFs were generated in
PyMOL using the NZF-1-F2 structure as a scaffold (19). These structures were compared
to the structures of WT NZF-1-F2 and the modelled structure of NZF-1-F3.
The
likelihood of H-bonding interactions between H and Y (for WT F2), Q and Y (for the F2
mutant, H515Q), H and R (for WT F3), or between Q and R (for the F3 mutant, H559Q)
were determined by estimating the bond distances between the potential hydrogen bond
donor and acceptor atoms (Figure 4.7). For F2 and H515Q, the H/Q likely serves as the
H-bond acceptor while Y serves as the H-bond donor. The closest calculated bond
distances for these constructs are 4.5 and 4.6 Å, respectively. These distances are too long
for a H-bond interaction to occur (20). For WT F3 and H559Q, where H/Q serves as the
H-bond donor and R as the acceptor, these distances are 2.8 and 3.1 Å, which are typical
distances for a hydrogen bonding interactions (20). This analysis suggests that hydrogen
bonding is important for the fold and function of F3, but not for F2. The DNA binding
results can be interpreted to support this conclusion. For F2, where hydrogen bonding
does not appear to be important for function, the H to Q mutation disrupted another key
feature that is important for fold and function, thus explaining the decrease in affinity of
the mutant for DNA. For F3, where H-bonding appears to be important, the mutation of
H to Q (H559Q) retains the hydrogen bond, but DNA binding is still negatively impacted.
This can be explained by the observation that Q does not completely mimic the hydrogen
bonding of the native H. The acceptor atoms of H and Q (N and O, respectively) are not
only in different orientations, but could be accepting the H-bond from different amides on
the donor arginine (Figure 4.7). Therefore, we propose that these differences in WT and
110
mutant F3 affect folding and DNA binding. Alternatively, Y520 and R564 could directly
contact DNA, thus any mutation would result in loss of binding.
Taken together, these results underscore the complexity of the DNA binding
interactions mediated by NZF-1. The published structures of single ZF domains contain
minimal secondary structure and how these structures change upon DNA binding is not
known. Attempts to obtain the structure of NZF-1-F2F3 bound to its DNA partner are
currently in progress in our lab to further unravel the complex zinc mediated NZF1/DNA recognition event.
111
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shuffling: mixing and matching cysteine and histidine residues in zinc finger
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Matthews, J. M., and Sunde, M. (2002) Zinc fingers--folds for many occasions,
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4.
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insights into structural and functional diversity, Curr. Opin. Struct. Biol. 11, 3946.
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Xia, Y. R., Lusis, A. J., and Rosenfeld, M. G. (1996) A novel family of Cys-Cys,
His-Cys zinc finger transcription factors expressed in developing nervous system
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7.
Kim, J. G., and Hudson, L. D. (1992) Novel member of the zinc finger
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8.
Yee, K. S., and Yu, V. C. (1998) Isolation and characterization of a novel member
of the neural zinc finger factor/myelin transcription factor family with
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9.
Jandrig, B., Seitz, S., Hinzmann, B., Arnold, W., Micheel, B., Koelble, K.,
Siebert, R., Schwartz, A., Ruecker, K., Schlag, P. M., Scherneck, S., and
Rosenthal, A. (2004) ST18 is a breast cancer tumor suppressor gene at human
chromosome 8q11.2, Oncogene 23, 9295-9302.
10.
Berkovits, H. J., and Berg, J. M. (1999) Metal and DNA binding properties of a
two-domain fragment of neural zinc finger factor 1, a CCHC-type zinc binding
protein, Biochemistry 38, 16826-16830.
11.
Besold, A. N., Oluyadi, A. A., and Michel, S. L. (2013) Switching metal ion
coordination and DNA Recognition in a Tandem CCHHC-type zinc finger
peptide, Inorganic chemistry 52, 4721-4728.
12.
Michael, S. F., Kilfoil, V. J., Schmidt, M. H., Amann, B. T., and Berg, J. M.
(1992) Metal binding and folding properties of a minimalist Cys2His2 zinc finger
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America 89, 4796-4800.
13.
Dhanasekaran, M., Negi, S., and Sugiura, Y. (2006) Designer zinc finger proteins:
tools for creating artificial DNA-binding functional proteins, Acc. Chem. Res. 39,
45-52.
14.
Gamsjaeger, R., Swanton, M. K., Kobus, F. J., Lehtomaki, E., Lowry, J. A.,
Kwan, A. H., Matthews, J. M., and Mackay, J. P. (2008) Structural and
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biophysical analysis of the DNA binding properties of myelin transcription factor
1, J. Biol. Chem. 283, 5158-5167.
15.
Berkovits-Cymet, H. J., Amann, B. T., and Berg, J. M. (2004) Solution structure
of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA
binding, Biochemistry 43, 898-903.
16.
Blasie, C. A., and Berg, J. M. (2000) Toward ligand identification within a
CCHHC zinc-binding domain from the NZF/MyT1 family, Inorganic chemistry
39, 348-351.
17.
Gamsjaeger, R., O'Connell, M. R., Cubeddu, L., Shepherd, N. E., Lowry, J. A.,
Kwan, A. H., Vandevenne, M., Swanton, M. K., Matthews, J. M., and Mackay, J.
P. (2013) A structural analysis of DNA binding by myelin transcription factor 1
double zinc fingers, J. Biol. Chem. 288, 35180-35191.
18.
Rich, A. M., Bombarda, E., Schenk, A. D., Lee, P. E., Cox, E. H., Spuches, A.
M., Hudson, L. D., Kieffer, B., and Wilcox, D. E. (2012) Thermodynamics of
Zn2+ binding to Cys2His2 and Cys2HisCys zinc fingers and a Cys4 transcription
factor site, J. Am. Chem. Soc. 134, 10405-10418.
19.
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Berg, J., Tymoczko, J. L., and Stryer, L. (2007) Biochemistry, 7 ed., W H
Freeman, New York, N.Y.
21.
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investigation of metalloproteins, Advances in inorganic biochemistry 6, 71-111.
114
22.
Berg, J. M., and Godwin, H. A. (1997) Lessons from zinc-binding peptides, Annu.
Rev. Biophys. Biomol. Struct. 26, 357-371.
23.
Berg, J. M., and Merkle, D. L. (1989) On the Metal-Ion Specificity of Zinc Finger
Proteins, J. Am. Chem. Soc. 111, 3759-3761.
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Functional characterization of iron-substituted neural zinc finger factor 1: metal
and DNA binding, JBIC, J. Biol. Inorg. Chem. 15, 583-590.
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binding to structural zinc-binding sites: accounting quantitatively for pH and
metal ion buffering effects, Analytical biochemistry 320, 39-54.
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Kang, P. C., Eaton, G. R., and Eaton, S. S. (1994) Puled Electron Paramagnetic
Resonance of High-Spin Cobalt(II) Complexes, Inorg. Chem. 33, 3660-3665.
27.
Makinen, M. W., Kuo, L. C., Yim, M. B., Wells, G. B., Fukuyama, J. M., and
Kim, J. E. (1985) Ground Term Splitting of High-Spin Co2+ as a probe of
Coordination Structure. 1. Dependence of the Splitting on Coordination
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28.
Kuo, L. C., and Makinen, M. W. (1985) Ground Term Splitting of High-Spin
Co2+ as a probe of Coordination Structure. 2. The Ligand Environment of the
Active Site Metal Ion of Carboxypeptidase A in Ester Hydrolysis
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29.
Walsby, C. J., Krepkiy, D., Petering, D. H., and Hoffman, B. M. (2003) Cobaltsubstituted zinc finger 3 of transcription factor IIIA: interactions with cognate
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DNA detected by (31)P ENDOR spectroscopy, J. Am. Chem. Soc. 125, 75027503.
30.
Dathan, N., Zaccaro, L., Esposito, S., Isernia, C., Omichinski, J. G., Riccio, A.,
Pedone, C., Di Blasio, B., Fattorusso, R., and Pedone, P. V. (2002) The
Arabidopsis SUPERMAN protein is able to specifically bind DNA through its
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Parraga, G., Horvath, S. J., Eisen, A., Taylor, W. E., Hood, L., Young, E. T., and
Klevit, R. E. (1988) Zinc-dependent structure of a single-finger domain of yeast
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116
Chapter 5
A Designed Zinc Finger Peptide with Hydrolytic Activity*
5.1 Introduction
It is estimated that one-third of all proteins are bound to metal ions (1-4). Zinc is
an essential metal in biology and is found in a number of different metalloproteins where
its role can range from purely structural in nature to aiding in catalysis (5-10). Proteins
that utilize zinc as a structural element are collectively called zinc finger (ZF) proteins
(11). ZF proteins utilize a combination of four cysteine and/or histidine ligands to
coordinate zinc(II) into a tetrahedral geometry and fold into a three-dimensional
structure. The best-studied ZFs are the ‘classical’ ZF proteins, which bind zinc(II) using a
CCHH motif and adopt a ββα fold upon zinc coordination (Figure 5.1) (6, 11-14). At
least fourteen other classes of ZFs are known, with each class delineated by the ligands
that coordinate zinc and the structure that the peptide adopts upon zinc(II) coordination
(14-19). Proteins that utilize zinc sites for a catalytic role typically coordinate zinc(II)
using three or four amino acid ligands, usually histidine, aspartate, glutamate and/or
cysteine. Importantly, these catalytic zinc centers usually contain one or more open
coordination sites that are absent in structural zinc proteins, and which allow for binding
of substrates and small molecule (e.g. H2O) co-factors to promote reactivity (20, 21).
*Adapted from Besold, A.N.; Widger, L.R.; Namuswe, F.; Michalek, J.L.; Michel, S.L.J.;
Goldberg, D.P. 2014. A Designed Zinc Finger Peptide with Hydrolytic Activity. In preparation
117
(a)
(b)
(c)
(d)
Figure 5.1 Consensus peptide-1 (CP-1) constructs (a). ExPASy output of all known
CCHH zinc sites. (b). The alignment of the amino acid sequences of CP-1 from 1991,
CP-1 from 2013, and modified CP-1 [named CP-1(CAHH)] (c). A cartoon figure
showing the positions of the amino acids of CP-1(CCHH)-2013. The zinc coordinating
residues are colored pink and the highly conserved amino acids are colored teal. (d). The
NMR structure of ZFP268, which is an example of a classical CCHH ZF protein. The
zinc coordinating residues are colored pink and the highly conserved amino acids are
colored teal. This figure was made in PyMol (v. 1.1), PDB ID: 2EMW.
There is an ongoing interest in constructing synthetic analogs of both structural
and catalytic metal sites in the form of small molecules and peptides (22). These
complexes, which are often more tractable than the native proteins that they mimic, allow
researchers to address fundamental chemical and biophysical questions, such as how the
binding of metals are connected to protein folding in structural sites, and how they
promote reactivity in catalytic sites (22).
ZF proteins typically contain well-defined, structural zinc binding domains that
make up smaller sections of the larger protein. Efforts to mimic these ZFs have thus
118
involved the preparation of short peptides that match the sequence of a specific zinc(II)
binding domain of a particular protein, or match a consensus sequence of the domain of
interest derived from a number of related ZF proteins (23-34). For example, Berg and coworkers recognized that the sequences of classical ZFs could be aligned to generate an
optimal or consensus sequence. A peptide with this consensus sequence was prepared and
named consensus peptide-1 (or CP-1) (35) and was shown to exhibit maximal zinc(II)
binding affinity and optimal protein folding (35-38).
Catalytic zinc sites in proteins have been modeled by a synthetic approach in
which organic ligands are designed to reproduce some of the key structural features of the
first coordination sphere found at the active site of the protein. For example, models of
carbonic anhydrase and alcohol dehydrogenase have been synthesized with various
polyamino, polypyrazolyl and polypyridyl ligands, and have yielded important insights
regarding the structure and function of the metal active sites (39). The hydrolytic enzyme
peptide deformylase (PDF) promotes the hydrolysis of N-terminal formyl groups of
newly synthesized peptides, and contains a divalent metal ion bound to the protein
through two His and one Cys residue (40, 41). The active site of this enzyme has been
modeled by some of us, with a key focus on the differences in reactivity observed for this
enzyme when bound to zinc(II) versus iron(II) (42-46). Our earlier studies, together with
the many other studies on synthetic analogs of catalytic zinc sites, have provided
information on mechanism and reaction intermediates that have helped gain an
understanding of structure/function relationships in zinc enzymes.
Although most efforts in modeling catalytic zinc sites have involved synthetic
small molecule analogs, there are a few examples in which peptides have been designed
119
to mimic catalytic zinc sites (47, 48). One early attempt by Berg and coworkers involved
modifying the structural ZF protein CP-1 by truncating the last four amino acids. This
truncation deleted one of the metal coordinating histidine ligands, which was predicted to
make the peptide hydrolytically active by providing an open site for water coordination.
Although zinc(II) was shown to coordinate to this peptide, it was not catalytically active
toward a chromogenic substrate, 4-nitrophenyl acetate (4-NA) (49). Subsequently,
Suguira and co-workers adopted a similar approach, involving the modification of a
structural ZF site, and successfully achieved catalytic hydrolysis activity. They designed
a number of catalytically active ZF peptides by modifying one of the ZF domains of the
ZF protein specificity protein 1 (Sp1), and some of these modified domains exhibited
hydrolytic activity toward carboxylic ester and circular DNA substrates (50-52).
Given the catalytic activity seen with re-engineered Sp1, we decided that CP-1
was worthy of further investigation for possible conversion to a hydrolytic enzyme, since
such a result would demonstrate that the most basic consensus sequence for a structural
ZF protein was capable of sustaining catalytic activity. Instead of removing a His ligand
as done previously (49), we sought to impart hydrolytic activity to CP-1 by incorporating
the ligand set of a known, hydrolytically active enzyme, PDF. In this report we describe
the modification of the ‘wild type’ CP-1(CCHH) through mutation of a coordinating
cysteine to a non-coordinating alanine, CP-1(CAHH), providing the N2S ligand set of
PDF. We demonstrate that this mutant, CP-1(CAHH), coordinates cobalt(II), likely in a
pentacoordinate geometry. Evidence for Zn(II) coordination to CP-1(CAHH) is also
presented and binding of these divalent metal ions is found to induce partial folding of
the peptide. It is shown that mutant CP-1(CAHH) exhibits good hydrolytic activity
120
toward 4-NA in both the metal bound and apo forms, and mechanistic hypotheses for the
observed hydrolytic reactivity are evaluated.
5.2 Materials and methods
5.2.1 Identification of consensus sequence for CCHH ZFs (CP-1 update)
To determine an updated consensus classical ZF sequence, a search of “zinc finger” using
PROSITE (prosite.expasy.org), which is part of the ExPASy suite of programs, was
performed (53, 54).
From this search, a document ‘PDOC00028,’ which contains
information about all CCHH-type ZF domains for which sequence information is
available, was selected. The weblogo for the updated consensus sequence, named CP1(CCHH)-2013 was generated via the ‘retrieve the sequence logo from the alignment’
link (55, 56). This sequence logo was constructed from 13,367 true positive hits from the
UniProtKB/Swiss-Prot databank and is shown in Figure 5.1a (57).
5.2.2 Peptide preparation
The CP-1(CAHH) peptide (PYKCPEAGKSFSQKSDLVKHQRTHTG) was purchased
from Biomatik (Wilmington, DE) and stored in the solid form at -20 °C until use. A small
amount of the solid peptide was resuspended in buffer prior to use and assayed for purity.
A single peak was observed at 20% acetonitrile:80% water via High Performance Liquid
Chromatography (HPLC). The identity of the isolated peak was confirmed by Matrix
Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF
MS); [observed: 2931.23 (M+); expected 2931.45]. The reduction state of the peptide was
121
determined by measuring the ratio of total peptide to Co(II)-peptide. Typically, >95% of
the peptide was reduced.
(58, 59) The peptide was stored and manipulated under
anaerobic conditions at all times (Coy Anaerobic Box, (95% N2, 5 % H2).
5.2.3 UV-visible metal binding titrations
Metal binding titrations were performed on a PerkinElmer Lambda 25 UV-visible
spectrometer. In a typical experiment, CoCl2 was titrated into a solution of apo-CP1(CAHH) (600 µM in 1 mL) dissolved in HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid) (100 mM) with NaCl (50 mM) at pH 7.5. The titration
included additions of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 5.0, 10, 15, and 20
molar equivalents of CoCl2. The shape of the spectrum did not change during the course
of the experiment, suggesting that Co(II) binds to the same site throughout the titration.
A plot of A630 versus CoCl2 was fit to a 1:1 binding model (equation 3) using non-linear
least squares analysis (KaleidaGraph, Synergy Software). An upper limit dissociation
constant (Kd) for cobalt(II) was determined.
(1)
(2)
(3) [PCo] =
P + Co
Kd =
PCo
[P][Co]
[PCo]
[P]total +[Co]total + K d - ([P]total +[Co]total + K d )2 - 4[P]total [Co]total
2[Co]total
122
Where P = Peptide, Co = Cobalt. The ability of Zn(II) to displace Co(II) was determined
by titrating Zn(II) into a solution of Co(II)-peptide (20:1 Co(II):peptide stoichiometry).
As Zn(II) was titrated, the Co(II) d-d bands disappeared, indicating that Zn(II) was
replacing Co(II) at the coordination site.
5.2.4 Nuclear magnetic resonance (NMR) spectroscopy
All NMR experiments were recorded on a 600 MHz Bruker spectrometer with the
temperature maintained at 25 °C. Apo-CP-1(CAHH) and Zn(II)-CP-1(CAHH) (350 µM)
were prepared in 25 mM deuterated Tris (tris(hydroxymethyl)aminomethane) containing
5% D2O at pH 7.5 and at pH 6.0. The Zn(II)-(CAHH) samples included 1 and 4
equivalents of Zn(II) at pH values of 7.5 and 6.0, respectively. All of the NMR spectral
data
were
processed
using
Spinworks
software
(http://www.umanitoba.ca./chemistry/nmr/spinworks).
5.2.5 Hydrolysis of 4-nitrophenyl acetate (4-NA)
The hydrolysis of 4-NA by the peptides was monitored using a PerkinElmer Lambda 25
UV-visible spectrometer by following the production of the reaction product, 4nitrophenolate (4-NP), at 400 nm. Each reaction was performed in 100 mM HEPES, 50
mM NaCl, pH 7.5 with 1% acetonitrile. 100 µM M-CP-1(CAHH) [M = Zn(II) or Co(II)]
(prepared at a 1:1 stoichiometry of M(II):peptide) was incubated with various
concentrations of 4-NA (dissolved in acetonitrile) and the absorbance at 400 nm was
recorded at 10 second intervals. The initial rate of hydrolysis was monitored up to a 5%
yield of the product, 4-NP. Background hydrolysis was measured by incubation of 4-NA
123
with either 100 µM ZnCl2 or 100 µM CoCl2 in the above buffer to determine if the metal
ions alone could promote hydrolysis. In both instances, a low level of hydrolysis was
recorded, and the appropriate spectrum for this hydrolysis was subtracted from the
spectrum of the corresponding M-CP-1(CAHH) reaction with 4-NA. The method of
initial rates was employed to monitor the reaction kinetics, and was then analyzed with
the kinetic model shown in eqs 4 and 5:
𝑣𝑖 = 𝑘 ′′ [M − CP − 1(CAHH)]0 [4 − NA]0
𝑣𝑖
[M−CP−1(CAHH)]0
= 𝑘 ′′ [4 − NA]0
(4)
(5)
where i = the initial rate of the reaction up to 5% conversion; [M-CP-1(CAHH)]0 =
initial concentration of metal peptide complex; [4-NA]0 = initial concentration of
substrate; k” = the second-order rate constant. The initial rate i was obtained from linear
fits of concentration vs time plots for 4-nitrophenolate (4-NP), where the concentration is
given by [4-NP] = A400/•l ( = 12,800 M-1 cm-1; l = 1-cm path length). Second-order rate
constants (k” values) were determined according to eq 5 by taking the slopes of the bestfit lines of i/[M-CP-1(CAHH)]0 vs [4-NA] (42, 44, 60). Apo-CP-1(CAHH) experiments
were performed in the manner described above, but the reaction was carried out under
anaerobic conditions in the absence of any metal ions to prevent cysteine oxidation. The
M(II)-CP-1(CAHH) experiments were performed in triplicate.
124
5.3 Results and discussion
5.3.1 Revisiting the consensus peptide
The ‘classical’ ZF proteins, which are the best-studied, contain domains with two
cysteine and two histidine ligands. The overall form is (Tyr,Phe)-X-Cys-X2,4-Cys-X3Phe-X5-Leu-X2-His-X3,4-His, with X equaling any amino acid (13). These proteins were
first identified in the mid-1980s, before any comprehensive genome sequencing of whole
organisms had been achieved (61-63). In 1991, Berg and co-workers aligned all of the
sequences of known ZFs, 131 sequences from 18 proteins, and identified a consensus
peptide
sequence
(CP-1)
with
the
sequence
ProTyrLysCysProGluCysGlyLysSerPheSerGlnLysSerAspLeuValLysHisGlnArgThrHis
ThrGly. This sequence was shown to bind cobalt(II) and zinc(II) more tightly (lower K d)
than any native ‘classical’ ZF. Thus, CP-1 is considered the optimized ‘classical’ ZF
(35). CP-1 has subsequently been utilized for a variety of studies aimed at understanding
topics ranging from the thermodynamics and kinetics of ZF protein folding, to the design
of new ZF-based complexes (30, 35-38, 64, 65). The most recent example is seen in the
work of Sénèque and Latour, where CP-1 and a series of mutants were utilized to
systematically measure binding affinities for Zn(II) and Co(II) ions by using competitive
chelators, and to determine the kinetics of metal ion exchange and speciation (38). Thus,
the original CP-1, determined from a small subset of sequenced proteins, remains
relevant to current work on ZF biochemistry.
An additional 13,236 CCHH ZF sequences have been identified since the initial
report of CP-1, as a result of the proliferation of sequenced genomes from high125
throughput proteomics efforts. The original CP-1 sequence was investigated here to
determine if the peptide that was designed in 1991 from only 131 sequences is still the
most conserved sequence. Using the EXPasy program, all of the CCHH type ZF domains
that have been sequenced and deposited in protein databases were extracted and aligned,
and a Prosite Sequence Logo was then generated (Figure 5.1a). A Sequence Logo is a
visualization of the sequence conservation between homologous proteins. The output of a
sequence logo shows a protein sequence with each amino acid represented at a specific
height, which corresponds to units of ‘bits’ on the y-axis. The height of the amino acid
indicates the frequency with which it occurs in that specific position throughout all of the
sequences analyzed. When the amino acid is conserved at the 100% level, the height of
the amino acid will be at its maximal level, which is 4 bits. When the amino acid is not
conserved 100% of the time, several amino acids will be placed in a given column in the
order of their percent conservation, with the most conserved amino acid appearing at the
top (56). Remarkably, a comparison of the new consensus sequence, CP-1(CCHH)-2013,
with the original consensus sequence, CP-1(CCHH)-1991, shows a high degree of
sequence conservation (Figure 5.1b). Despite the small number of ZF sequences (less
than 1% of those known today) analyzed by Berg and co-workers over twenty years ago,
their original CP-1 sequence remains an accurate representation of a classical ZF domain.
Thus the use of CP-1 was validated as a scaffold for the work described herein.
5.3.2 Modifying CP-1 to promote hydrolytic activity
Previous work from Berg attempted to make a mutation at the metal binding site of
CP-1 to convert the structural zinc(II) site into a site capable of mediating hydrolysis
126
(49). CP-1 was truncated such that the last four amino acids were removed, including
one of the histidine ligands, with the goal of opening up a site on the metal center for the
binding and activation of H2O. The activation of H2O at Zn(II) in hydrolytic zinc
enzymes, in which the pKa of the bound H2O molecule is lowered, is a key part of their
function and provides access to a nucleophilic Zn-OH species (20, 21). No hydrolytic
activity was observed in this earlier study, despite the successful formation of the HCC
(NS2) zinc center and the generation of an open coordination site at the zinc(II) ion in the
CP-1(HCC) mutant.
Some of us have previously constructed hydrolytically active, synthetic (smallmolecule) analogs of the active site of peptide deformylase (PDF). This enzyme catalyzes
the hydrolytic deformylation of the N-terminus of newly synthesized polypeptides. PDF
is activated by the coordination of a divalent metal ion [Zn(II), Fe(II), or Co(II)] to one
cysteine and two histidine residues (N2S donor set) in the active site (40, 41, 66). The
activation of H2O occurs at the metal center, ultimately leading to the hydrolysis of
formylated substrates. We speculated that mutation of one of the Cys ligands in CP-1 to a
non-coordinating residue would convert the metal site to an N2S-MII center and provide a
mimic of the PDF active site that would have a higher Lewis acidity than the early CP1(HCC) mutant. The Lewis acidities will directly influence the extent of water activation
and the generation of the hydrolytically active zinc(II)-hydroxide species for CP-1(HCC)
and CP-1(CAHH) according to equations 6 and 7, respectively.
127
0
OH2
SCys
SCys
SCys
NHis
ZnII
NHis
OH
SCys
SCys
NHis
OH2
OH
ZnII
ZnII
NHis
SCys
NHis
H+
ZnII
(6)
0
H+
NHis
(7)
5.3.3 CP-1(CAHH) and coordination of Co(II)
CP-1 was modified to mimic the HHC metal binding motif of the active site of PDF. The
native protein, referred to as CP-1(CCHH), was altered such that the second coordinating
cysteine residue was mutated to a non-coordinating alanine residue [CP-1(CAHH)]
(Figure 5.1b). Initial attempts to synthesize this peptide independently by using a
Symphony Quartet peptide synthesizer proved problematic. A significant portion of the
peptide was not synthesized to completion, and separation of the incompletely
synthesized peptide from the fully intact peptide via HPLC proved prohibitive due to low
yields. The peptide was then purchased from Bio-synthesis (Lewisville, TX) in the crude
form and purified further in our laboratory by HPLC. This peptide was not soluble in the
presence of metal ions. Pure, stable peptide was obtained from Biomatik (Wilmington,
DE). The purity of the peptide was independently measured in our laboratory via
analytical HPLC and found to be >95% pure. This peptide was utilized without further
purification. We therefore note that the preparation of mutant CP-1 is not straightforward.
128
Interestingly, Sénèque and Latour report similar difficulties in the preparation of CP-1 in
their recent work (38).
Initial experiments focused on determining the influence of the Cys-to-Ala
mutation on the metal-binding properties of the peptide. The ability of CP-1(CAHH) to
coordinate Zn(II) was determined indirectly by using Co(II) as a spectroscopic probe for
Zn(II). This approach takes advantage of the rich spectroscopic properties of Co(II) in a
tetrahedral coordination environment with sulfur and nitrogen ligands. The Co(II) (d7) ion
exhibits distinct d-d transitions between 550 – 750 nm that can be correlated with 4coordinate, 5-coordinate, and 6-coordinate ligand environments around the metal. In
addition, when sulfur, and to a lesser extent nitrogen, serve as ligands, ligand-to-metal
charge transfer (LMCT) bands are often observed in the near-UV region (67-69).
Addition of CoCl2 to CP-1(CAHH) at pH 7.5 leads to a spectrum that exhibits
(a)
(b)
(c)
Figure 5.2 Comparison of UV-Visible Spectra of CCHH vs CAHH CP-1 (a). sequence
alignment of CP-1(CCHH)Q∆W with CP-1-(CAHH). (b). UV-visible spectra of Co(II)CP-1(CCHH) Q∆W. C. UV-visible spectra of Co(II)-CP-1(CAHH).
129
bands with maxima at 320, 530, and 630 nm (Figure 5.2b). For comparison, we measured
the UV-visible spectrum of Co(II) bound to a modified version of CP-1(CCHH) (Figure
5.2a) . This modified version, CP-1(CCHH)QW, which was created to investigate
metal ion binding using fluorescence titrations in further experiments, contains a
tryptophan residue in place of a glutamine as well as two additional amino acids at the C
terminus (Figure 5.2a). The Co(II)-CP-1(CCHH)QW UV-visible spectrum is identical
to that of Co(II)-CP-1(CCHH)-1991 (35). The d-d bands for Co(II)-CP-1(CCHH)QW
and Co(II)-CP-1(CCHH)-1991 are red shifted with maxima at 570 nm and 645 nm
compared to CP-1(CAHH) (Figure 5.2b). The band at 320 nm remains the same. The
position of the d-d transitions in relation to the coordination environment of the cobalt(II)
ion has been well studied. It is known that cobalt(II) in a 4-coordinate, tetrahedral
coordination geometry exhibits absorption maxima at 625 ± 50 nm, while cobalt(II) in a
6-coordinate, octahedral geometry gives maxima at 525 ± 50 nm. Cobalt(II) in a
pentacoordinate geometry has maxima between these values (50, 67). While both the
CCHH and CAHH CP-1 constructs exhibit absorbance maxima that best fit for
tetrahedral coordination at cobalt(II), the absorbance bands for CP-1(CAHH) are blue
shifted when compared to CP-1(CCHH), suggesting that the geometry may be distorted
from an ideal tetrahedral environment to a 5-coordinate geometry. In addition, the
extinction coefficient (ε) at the absorption maxima provides information about
coordination number. Typically, tetrahedral complexes have an ε greater than 300 M -1
cm-1, while octahedral complexes have an ε below 30 M-1 cm-1, with the ε values for
pentacoordinate complexes again falling somewhere in between 4- and 6-coordinate
complexes ( 50 ≤ ε ≤ 250 M-1 cm-1) (50, 67). At its maxima, CP-1(CCHH) has an
130
extinction coefficient of 510 M-1 cm-1, suggestive of tetrahedral coordination. CP1(CAHH) has a significantly lower extinction coefficient of 60 M-1 cm-1, which is more
consistent with a pentacoordinate (or even an octahedral) environment. Thus, in addition
to the cysteine and two histidine residues, Co(II) [and, by inference, Zn(II)], is likely
coordinated by one or two water molecules.
There is precedence for water coordination to zinc ions in re-engineered structural
zinc sites. For example, when the second coordinating cysteine from a de novo four-helix
bundle peptide called Zα4 that contained four pre-organized zinc binding ligands
(Cys2His2) was mutated to an alanine, the Co(II) UV-visible spectrum was altered: the
max for the cobalt(II) d-d bands was blue-shifted to 592 nm from 617 nm observed for
unmodified Zα4 and the extinction coefficient was decreased to ε = 213 M-1 cm-1 from ε
= 390 M-1 cm-1 (70). These data are suggestive of 5-coordinate geometry at the metal site,
with two exogenous water molecules serving as ligands, in addition to the cysteine and
histidine residues from the Zα4 ligand (70). Similarly, modification of the first
coordinating cysteine of Zα4 to an alanine also altered the absorption spectrum, with a
measured extinction coefficient for the Co(II) d-d bands of ε = 60 M-1cm-1 (the max was
not reported).
Similarly, Nomura and Sugiura found that when they systematically
mutated metal coordinating cysteine and histidine ligands of the second ZF domain of
Sp1 to non-coordinating ligands (alanine or glycine), the resultant metal site gave Co(II)
UV-vis spectra consistent with a pentacoordinate geometry. The ligand sets of the Sp1
peptides were CCHG, CCAH, and CCGH respectively, and extinction coefficients (ε) of
191 M-1cm-1, 165 M-1cm-1, and 135 M-1cm-1 at 655, 620 and 625 nm for Co(II)
coordination were reported (50, 51). There are also examples of these types of metal
131
(b)
(a)
Figure 5.3 Co(II) Titration of CP-1(CAHH) (a). Plot of the change in the absorption
spectrum between 250-800 nm as 600 µM CP-1(CAHH) is titrated with CoCl2. All
spectrophotometric experiments were performed in 100 mM HEPES, 50 mM NaCl at
pH 7.5 (b). Plot of absorbance at 630 nm versus the concentration of Co(II)Cl2 added
to CP-1(CAHH). The data were fit to a 1:1 binding equilibrium to yield an upper limit
dissociation constant of 170 µM. The solid line represents a nonlinear least-squares fit to
the 1:1 binding model.
centers in naturally occurring zinc enzymes. For example, when Co(II) is bound to the
zinc enzyme farnesyltransferase (FTase), the UV-vis spectrum exhibits a max at 560 nm,
with an extinction coefficient of 140 M-1 cm-1, which is indicative of a 5-coordinate
metal center with at least one water molecule bound to the metal (71).
In addition to creating an open coordination site to which water can bind, it is not
surprising that mutations that alter the metal binding site in ZFs have also been shown to
affect the ZF’s affinity for the metal ion. For instance, Klemba and Regan demonstrated
that mutagenesis of the peptide Zα4 to favor pentacoordinate geometry at the metal site
resulted in a 33-fold decrease in Co(II) ion binding affinity when compared to the Co(II)
binding to the wild type Zα4 peptide (70).
We thus performed spectroscopic titrations of CP-1(CAHH) with cobalt(II) ions
to determine the effect of the alanine mutation on the affinity for metal ions (Figure
132
5.3a). In this approach, the apo-peptide was titrated with cobalt(II) dichloride at pH 7.5 in
100 mM HEPES (50 mM NaCl) buffer until saturation was reached, as monitored by the
appearance of the d-d transitions in the visible region. The resulting titration curve
(Abs630
nm
versus [Co(II)], Figure 5.3b) was easily fit to a 1:1 binding equilibrium,
consistent with the formation of a 1:1 cobalt/peptide complex. The best fit of the data to
eq 1-3 led to the determination of an upper limit for the dissociation constant (Kd) of 170
µM for cobalt(II) ion binding to CP-1(CAHH). In comparison, a Kd of 50 nM has been
reported for CP-1(CCHH) when measured in the same manner while a Kapparent of 10-15 M
is reported when a more rigorous, competitive titration is performed (38). Thus, the single
Ala mutation in CP-1(CAHH) leads to a significant weakening of the binding affinity for
Co(II) ion, CP-1(CCHH), but this result is not surprising given that one of the peptidederived metal-binding ligands has been removed. Despite the deletion of one of the
metal-binding Cys donors, the Kd for CP-1(CAHH) is still within the regime of
dissociation constants that have been reported for cobalt(II) coordination to ZF proteins
(31, 38, 72).
5.3.4 Zn(II) coordination of CP-1(CAHH)
The cobalt(II) ion was next used as a spectroscopic probe for measuring the
binding of zinc(II) to CP-1(CAHH), which is spectroscopically silent. This strategy is
well established for measuring zinc(II) binding affinities for ZF proteins (27, 31, 32, 58,
68, 73-75). A solution of fully loaded Co(II)-CP-1(CAHH) was prepared by combining
the peptide with excess Co(II) ions (20 equiv of CoCl2) at pH 7.5. Spectroscopic titrations
were performed by the addition of successive aliquots of ZnCl2, leading to the
133
progressive loss of cobalt(II) d-d transitions at 630 and 550 nm. A plot of A630 nm versus
[ZnCl2] is shown in Figure 5.4a. The sharp decrease in the absorbance for the Co(II)
marker band at 630 nm is indicative of displacement of Co(II) by Zn(II), confirming the
coordination of Zn(II) by CP-1(CAHH). The lack of curvature in this plot indicates that
the Zn(II) ion is binding very tightly in this concentration range, and the Kd for Zn(II) is
too large to be measured with these data. These results for Zn(II) ion coordination to CP1(CAHH) as compared to the Co(II) ion fall in line with other ZF proteins, in which the
binding of Zn(II) is thermodynamically favored over that of Co(II) (27, 29-32, 35, 36, 38,
67, 72, 75-79).
134
5.3.5 Secondary structure of CP-1(CAHH) upon metal ion coordination
The binding of zinc(II) ion to ZFs can be expected to induce a structural change in
the protein. ZF domains in the apo form are usually unstructured, only adopting
secondary structure upon the coordination of a metal ion [e.g. Zn(II), Co(II)] (69, 80).
The chemical shift dispersion of amide N-H resonances can be correlated with the
presence of secondary structural elements in small peptides and proteins (81, 82), and the
folding of CP-1(CAHH) was examined by 1-D (1H) NMR spectroscopy in the presence
(b)
(a)
(c)
(d)
(e)
Figure 5.4 Zn(II) Binding of CP-1(CAHH) (a). Plot of absorbance at 630 nm versus the
concentration of ZnCl2 added to a saturated solution of Co(II)-CP-1(CAHH) [20:1
Co(II):CP-1(CAHH)]. All spectrophotometric experiments were performed in 100 mM
HEPES, 50 mM NaCl at pH 7.5 (b). 1H-NMR spectrum of apo-CP-1(CAHH) at pH 7.5, (c).
1H-NMR spectrum of apo-CP-1(CAHH) with 1 equivalent of ZnCl2 at pH 7.5, (d). 1H-NMR
spectrum of apo-CP-1(CAHH) at pH 6, (e). 1H-NMR spectrum of apo-CP-1(CAHH) with 4
equivalents of ZnCl2 at pH 6. The peptides for all NMR experiments were at a concentration
of 350 µM in 600 µL of 25 mM deuterated Tris with 5% D2O.
135
and absence of zinc(II) ions. The 1-D (1H) NMR spectrum for apo-CP-1(CAHH) is
shown in Figure 5.4b. The amide N-H peaks are found in the region 6.5 – 8.2 ppm and
are not well dispersed, as expected for an unstructured peptide. The addition of one
equivalent of ZnCl2 to apo-CP-1(CAHH) at pH 7.5 resulted in shifts in the aromatic
region, which are likely due to Zn(II) binding to the histidine ligands and some amide
proton dispersion (Figure 5.4c). It is well known that a slight lowering of the pH is often
necessary to induce good dispersion in the amide N-H region associated with protein
folding (83). Additional dispersion of the amide N-H resonances was observed between
7.2 – 8.2 ppm upon lowering the pH to 6.0 and adding a slight excess of Zn(II) ions (4
equiv), as seen by comparison of the spectra for apo-CP-1(CAHH) and Zn-CP-1(CAHH)
at pH 6.0 (Figure 5.4d-e). Similar experiments have been performed with CP-1(CCHH)
O
O
NO2
Zn(II)-CP-1(CAHH)
H2O/CH3CN
100 mM HEPES
50 mM NaCl
pH 7.5
O
O
O
NO2
Figure 5.5 Hydrolysis of 4-NA. A plot of the increase in the absorbance at 400 nM as
100 µM Zn(II)-CP-1(CAHH) reacts with 100 µM 4-NA to yield the chromogenic
product 4-NP.
136
and the peaks corresponding to the amide N-H protons were dispersed over a wider range
(6.5 – 9.4 ppm) upon metal ion coordination at pH 6.5 (38, 68). The NMR data indicate
that CP-1(CAHH) clearly coordinates zinc(II) ions at pH 6.0 and exhibits some
secondary structure formation upon metal ion binding, but does not fold to the same
extent as CP-1(CCHH).
5.3.6 Hydrolysis of 4-nitrophenyl acetate (4-NA)
The ability of CP-1(CAHH) to mediate hydrolysis in both the presence and
absence of divalent metal ions was examined with the test substrate 4-nitrophenyl acetate
(4-NA). The 4-NA substrate has been commonly used to measure the hydrolytic
efficiency of both zinc complexes as well as zinc peptides and proteins (44, 47, 84-89),
allowing for a direct comparison of the reactivity of CP-1(CAHH) against other
Figure 5.6 Formation of 4-NP over Time. A plot of 4-NP concentration
(A400/(ε•l)) as a function of time indicating the production of 4-NP in the reaction of
100 µM 4-NA with apo-CP-1(CAHH) (in red), Co(II)-CP-1(CAHH) (in blue), or
Zn(II)-CP-1(CAHH) (in pink), all at 100 µM . All data were collected at pH 7.5 in
100 mM HEPES, 50 mM NaCl at 25 °C.
137
hydrolytically active zinc complexes. The M-CP-1(CAHH) [M = Zn(II) or Co(II)]
peptide was incubated with 4-NA at pH 7.5 in 100 mM HEPES (1% acetonitrile, 50 mM
NaCl) buffer and the hydrolysis reaction was monitored by UV-visible spectroscopy. All
reactions were performed at 100 M M-CP-1(CAHH). Under these conditions, and with
a predicted upper limit Kd of 1 nM for Zn(II) binding to CP-1(CAHH), Zn(II)-CP1(CAHH) is the predominant species in solution (at a concentration of 99.68 M).
However, Co(II)-CP-1-(CAHH) has an upper limit Kd of 170  indicating that both
Co(II)-CP-1(CAHH) and apo-CP-1(CAHH) are present in solution (29 and 71 M),
respectively. The desired reaction gives the expected product, 4-nitrophenolate (4-NP),
which exhibits a characteristic absorbance at max = 400 nm ( = 12800 M-1 cm-1 at pH
7.5), appearing over time (Figure 5.5). An initial rate method was employed to monitor
the kinetics of hydrolysis of 4-NA. Initial rates (i) were obtained from the best-fit lines
Figure 5.7 Second Order Rate Constants of the Reaction with 4-NA Plot of i/[MCP-1(CAHH)] as a function of [4-NA] for the reaction of 4-NA with Co(II)-CP1(CAHH) (blue line) and Zn(II)-CP-1(CAHH) (pink line). All data were collected
at pH 7.5, 100 mM HEPES, 50 mM NaCl, 25 °C.
138
Table 5.1 Second order rate constants (k'') of the hydrolysis of 4-NA by various
complexes and peptides.
Complex
(PATH)ZnOH
k'' (M-1 s-1)
0.089 ± 0003
Ref
42
([12]aneN3)ZnOH
(cyclen)ZnOH
(CH3cyclen)ZnOH
([15]aneN3O2)ZnOH
ZnL1
ZnL3
ZnL8
Protein/Peptide
(His94Cys)CAII
Zn(II)-CCAH
Zn(II)-CCHA
Zn(II)-CGHH
Zn(II)-AHHH
Zn(II)-HHAH
Zn(II)-HHHH
apo-CGHH
Zn(II)-CP-1(CAHH)
Co(II)-CP-1(CAHH)
0.036 ± 0.003
0.1 ± 0.01
0.047 ± 0.001
83
84
85
81
86
86
86
Ref
82
51
51
51
51
51
51
51
This work
This work
0.6 ± 0.06
0.3908 ± 0.1
0.2791 ± 0.02
0.3863 ± 0.02
k'' (M-1 s-1)
117 ± 20
0.232 ± 0.0051
0.568 ± 0.0228
0.458 ± 0.0021
0.478 ± 0.0057
0.370 ± 0.0289
0.966 ± 0.0492
0.376 ± 0.0235
0.482 ± 0.034
0.556 ± 0.072
of [4-NP] versus time plots (Figure 5.6). An initial rate method was selected because of
the relatively slow rates observed for these reactions. The kinetics were measured over a
range of substrate concentrations (0.5 – 2 equiv vs [M-CP-1(CAHH)]), and plots of
i/[M-CP-1(CAHH)]0 versus [4-NA] for both Zn(II)- and Co(II)-CP-1(CAHH) are shown
in Figure 5.7. The best-fit lines in Figure 5.7 yield the second-order rate constants k” =
0.556 ± 0.072 M-1 s-1 and 0.482 ± 0.034 M-1 s-1 for the Co(II) and Zn(II) peptides,
respectively (eq 5). These rate constants are on the high end of those reported for many
zinc complexes, which mostly range from 0.036 – 0.6 M-1s-1 (Table 4.1) (44, 84, 86-89).
The rates are also faster than most of those seen for the hydrolytic activity of a series of
related mutant peptides based on the ZF domain of Sp1 reported by Nomura and Sugiura
(51), in which the metal-binding ligands were systematically varied (Table 4.1) and the
hydrolytic cleavage of 4-NA was examined. CP-1(CAHH) exhibits rates that are only
139
modestly slower than even the most active of the Sp1 peptides, which contains an HHHH
metal-binding motif. The zf-(HHHH) has a second-order rate constant of approximately
only a 2-fold greater increase than CP-1(CAHH) (51). Taken together, these data suggest
that the peptide environment of CP-1(CAHH) activates divalent metal ions toward
hydrolysis as well, or better than, many of the small-molecule organic ligands that have
been prepared for hydrolytic catalysis, and is at most only a factor of 5 slower than the
most hydrolytically active ZF peptides that mediate the hydrolysis of 4-NA.
The ability of apo-CP-1(CAHH) to hydrolyze 4-NA was also examined. The apo
form of the peptide does mediate the hydrolysis of 4-NA. The rate for apo-CP-1-(CAHH)
was the fastest, followed by Co(II)-CP-1-(CAHH) and then Zn(II)-CP-1-(CAHH). Under
the experimental conditions, Co(II)-CP-1-(CAHH), is present in both the apo- and Co-(II)
bound forms while Zn(II)-CP-1-(CAHH) is present as just the Zn(II) bound form. Thus,
the increase in rate for Co(II)-CP-1-(CAHH) suggest that both apo-CP-1-CAHH and
Co(II)-CP-1-CAHH are involved in the hydrolysis reaction (Figure 5.6). On the other
hand, the significant hydrolytic activity in the presence of Zn(II) ion can be attributed
mostly to the metal-bound peptide, as opposed to apo-peptide, because of the
predominance (>99%) of the zinc-bound form under the reaction conditions. This result
is in line with the previous findings from Nomura and Sugiura, in which the apo forms of
the mutant ZF domains of Sp1 exhibited similar hydrolytic activity toward 4-NA as their
metallated analogs (Table 5.1) (51). The ability of apo-peptide to hydrolyze 4-NA in the
former study was shown to track with the number of histidine residues, in contrast to the
Zn(II) complexes of the Sp1 mutants (51). Thus the hydrolytic activity of apo-CP1(CAHH) can be attributed to the inherent reactivity of the His residues, whereas the
140
slightly less active metal-bound CP-1(CAHH) peptides have His sites blocked by metal
ion coordination. These results strongly suggest that both Zn(II)- and Co(II)-CP1(CAHH) exhibit metal-mediated hydrolytic activity, which is likely controlled by the
nature of the ligands, the local structure of the metal site, and the overall folding of the
peptide.
The finding that the CP-1(CAHH) mutant exhibits similar activity towards 4-NA
as the Sp1(CGHH) mutant reported by Sugiura and co-workers is somewhat surprising.
CP-1 is an optimized Cys2His2 ZF, and its property of binding Zn(II) and Co(II) with
higher affinities than native Cys2His2 ZF type ZFs (such as Sp1) has been attributed to
the optimized amino acid sequence of CP-1. Based upon this property, one would predict
that the when CP-1 is modified at a single amino acid to allow for reactivity towards
substrates, the reactivity would be faster than the analogously modified Sp-1, because the
amino acid sequence of CP-1 has been optimized. The results presented here for CP-1CAHH do not support this prediction. Thus, there are limits to the benefits of optimizing
the sequence of a zinc finger protein.
5.4 Conclusions
We have shown that the consensus peptide for a classical ZF domain, CP-1, can
be modified by rational design to convert the structural zinc site into a zinc site capable of
efficiently mediating the hydrolysis of an external substrate. The single mutation of Cysto-Ala at the metal-binding site was made to give a CHH binding motif (CP-1(CAHH)),
based on the known ligand set for the hydrolytically active Zn(II)/Fe(II) enzyme peptide
deformylase. Despite the removal of one of the key metal binding ligands, CP-1(CAHH)
141
is still capable of binding Co(II) and Zn(II) with good affinity, and shows some
secondary structure formation upon binding metal ion, albeit less structurally ordered
than the native CP-1(CCHH) peptide. The hydrolytic cleavage of the test substrate 4-NA
mediated by M(II)-CP-1(CAHH) (M = Co, Zn) is faster than many of the known zinc
model complexes that have been designed as catalytic mimics for zinc enzymes. We have
thus shown that the CP-1 peptide represents a potential scaffold from which to create
efficient, catalytically active hydrolysis enzymes that utilize biomimetic divalent metal
ions. For high catalytic activity to be achieved, the non-metal coordinating amino acids
may need to be optimized to promote hydrolysis at the zinc site as well as provide the
proper structure to the catalytic domain. A systematic tuning of the metal active site as
well as the peptide structure by varying residues through a combinatorial and/or rational
approach may lead to such optimization.
142
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Kimura, E., Nakamura, I., Koike, T., Shionoya, M., Kodama, Y., Ikeda, T., and
Shiro, M. (1994) Carboxyester Hydrolysis Promoted by a New Zinc(Ii)
Macrocyclic Triamine Complex with an Alkoxide Pendant - a Model Study for
the Serine Alkoxide Nucleophile in Zinc Enzymes, J. Am. Chem. Soc. 116, 47644771.
153
87.
Koike, T., Takamura, M., and Kimura, E. (1994) Role of Zinc(Ii) in BetaLactamase-Ii - a Model Study with a Zinc(Ii) Macrocyclic Tetraamine (1,4,7,10Tetraazacyclododecane, Cyclen) Complex, J. Am. Chem. Soc. 116, 8443-8449.
88.
Koike, T., Kajitani, S., Nakamura, I., Kimura, E., and Shiro, M. (1995) The
Catalytic Carboxyester Hydrolysis by a New Zinc(Ii) Complex with an AlcoholPendant Cyclen (1-(2-Hydroxyethyl)-1,4,7,10-Tetraazacyclododecane) - a Novel
Model for Indirect Activation of the Serine Nucleophile by Zinc(Ii) in Zinc
Enzymes, J. Am. Chem. Soc. 117, 1210-1219.
89.
Subat, M., Woinaroschy, K., Anthofer, S., Malterer, B., and Konig, B. (2007)
1,4,7,10-tetraazacyclododecane metal complexes as potent promoters of
carboxyester hydrolysis under physiological conditions, Inorg. Chem. 46, 43364356.
154
Chapter 6
Engineering a Zinc Finger Protein as an Artificial RNA Cleaving Agent:
Tristetraprolin-Ribonuclease 4 (TTP2D-RNase4)*
6.1 Introduction
Tristetraprolin (TTP) is a non-classical CCCH-type zinc finger (ZF) protein that is
essential in controlling the inflammatory response (1-7). The role of TTP was discovered
using TTP-deficient mice, which exhibited a variety of inflammatory symptoms
including arthritis, cachexia, and conjunctivitis (6). Increased levels of the proinflammatory protein Tumor Necrosis Factor α (TNFα) were observed in these mice and
it was discovered that TTP plays a role in regulating the levels of this cytokine (5, 6).
Subsequently, TTP was shown to control the levels of a large number of other cytokines
including several interleukins (IL-2, IL-3, IL-6, IL-10, and IL-23) and interferon-γ (8-13).
In addition, TTP also regulates enzymes associated with inflammation, including
cyclooxygenase-2, which generates prostaglandins and is upregulated in inflammation,
and inducible nitric oxide synthase, which generates NO in inflammatory cells (14-17).
Controlling these inflammatory pathways is essential as various disorders, such as
rheumatoid arthritis and inflammatory bowel disease, are results of an aberrant
inflammatory pathway (18). Chronic inflammation has also been linked to cancer, a
disease in which TTP plays an important role (18-20).
*This work was done in collaboration with Dr. Gerald Wilson’s laboratory.
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Figure 6.1 NMR Solution Structure of Tis11d, a TTP Homolog. Tis11d
(purple), a homolog of TTP, is shown interaction with AU-rich RNA (blue).
(PDBID 1RGO)
Initial evidence for TTP’s role in cancer came from studies examining a mast cell
model that over-expresses IL-3 (21). Expression of TTP in these cells resulted in
decreased IL-3, as TTP regulates this cytokine. Injection of this cell line transfected with
TTP into mice led to slowed tumor growth and impaired vascularization of these tumors
(21). Since this initial discovery, TTP has been shown to be downregulated in several
types of cancers and has been linked to poor outcomes in both breast and prostate cancers
(22-24). TTP exhibits some these anti-cancer effects by regulating expression of various
tumor-promoting proteins, such as vascular endothelial growth factor, cyclin D1,
hypoxia-inducible factor 1α, matrix metalloproteinase-1, and urokinase plasminogen
activator (25-29). Thus, not only is TTP essential in regulating the inflammatory
response, it also has the potential to function as a potent anti-cancer agent.
TTP functions to regulate these myriad of proteins at the mRNA level.
Specifically, TTP recognizes AU-rich sequence elements (AREs) present in the
3’untranslated region (UTR) of the mRNA (30). It is estimated that approximately 8% of
156
(a)
(b)
Figure 6.2 ZF Domains of TTP (a) NMR solution structure of the N terminal ZF of
TTP (PDBID 1M9O) (b) Amino acid sequence of TTP-2D. Zn(II) coordinating residues
shown in pink
all RNAs contain these AREs, which are essential for mRNA decay (30, 31). The
protein/RNA interaction of TTP involves two CCCH ZF domains which selectively bind
AREs that contain UUAUUUAUU or similar motifs (Figure 6.1 and 6.2) (3, 32-41).
Binding to this sequence marks these RNAs for degradation via recruitment of decapping
enzymes, 3’ and 5’ exonuclease activities, and nucleating P body formation (33, 42, 43).
Given TTP’s wide range of RNA targets that make this protein important for
controlling both the inflammatory response and cancerous growth, we hypothesize that
enhancing the activity of TTP could be a novel strategy to turn off inflammation and
tumorigenesis. Thus, we sought to develop a TTP construct that could be utilized in such
a manner. The introduction of full-length TTP to target inflammatory or cancer cells is
not an ideal therapeutic mechanism, as full-length TTP is subject to phosphorylation
which deactivates this protein (44, 45). However, efforts in the Michel lab have produced
a construct of TTP that contains just the two CCCH ZF domains which are involved in
RNA recognition, termed TTP2D (Figure 6.2) (40). TTP2D is free of the phosphorylation
sites that render full length TTP inactive making it a good candidate for our strategy to
157
create a macromolecule to turn off inflammation. This construct is predicted to have
limited utility if delivered alone, however, because TTP lacks the ability to hydrolyze
mRNAs and thus relies on intracellular machinery to promote RNA degradation.
However, we reasoned that if this macromolecule is appended to an RNA cleavage
domain (e.g. a ribonuclease), the resultant chimeric protein would have the potential to
both target and cleave mRNA. This approach has strong precedence in ZF nucleases
(ZFN).
ZFNs are composed of a classical CCHH ZF DNA binding domain, which binds
specifically to DNA, and an endonuclease domain, which cleaves DNA (Figure 6.3) (4649). Upon dimerization at the target genes, these proteins create a double stranded DNA
break, which can result in mutagenesis of an undesirable gene via Non-Homologous End
Joining (NHEJ) or repair of a mutated gene via homology directed repair (HDR), if a
donor DNA strand is present (50). ZFNs are highly successful and have been used in
clinical trials for the treatment of glioblastoma and HIV (50, 51).
Figure 6.3 Representation of ZFN. ZFN consist of two domains. The ZF domain,
shown in teal with the Zn(II) represented in gray, and a FokI nuclease domain,
represented in red. The ZF domain, demonstrated here using the classical CCHH Xfin
domain represented three times (PDBID 1ZNF), binds spefically to DNA and the FokI
domain cleaves this DNA upon dimerization. Figure generated in Pymol (Xfin),
Microsoft Powerpoint (FokI), and ChemDraw (DNA).
158
Moreover, we believe that our strategy of creating a chimeric TTP-ribonuclease,
based upon ZFNs, offers advantages over current methods employed to regulate RNA.
These methods include RNA interference (RNAi), such as small interfering RNA
(siRNA) or short hairpin RNA (shRNA), as well as the CRISPR-based methods. siRNA
and shRNA work by promoting the degradation of known, complementary, RNA target
sequences, with shRNA being the longer acting method of RNAi (52). CRISPR-based
methods use a guide RNA that is targeted to complementary nucleotides. These guide
RNAs will recruit an endonuclease to cleave the target of interest using a double strand
break (53, 54). Both methods require targeting to a specific sequence and rely on
intracellular machinery for function. We propose that coupling TTP2D to a ribonuclease
offers a more beneficial means of controlling inflammation and cancer as doing so would
free TTP of its reliance on intracellular machinery that is needed to degrade RNA targets
and would allow for high catalytic turnover if provided with the proper nuclease. Finally,
Figure 6.4 Model of TTP Ribonuclease Strategy. TTP2D (pink) is
connected to RNase4 (teal) by a (G4S)3 linker sequence.
159
there is some recent and exciting precedence for our chimeric protein strategy. Wang and
co-workers successfully achieved specific cleavage of CUG repeats by linking the PUF
RNA binding domain of a protein named PIM to the PIN RNA cleavage domain of
SMG6 (55).
Here, we sought to create the first generation TTP ribonuclease and obtain proof
of principle data that this chimera can selectively bind and cleave ARE mRNA
sequences. We created a chimeric protein composed of the RNA binding ZF domains of
TTP (TTP2D) and ribonuclease 4 (RNase4) (Figure 6.4). RNase4 is the smallest member
of the RNase family of proteins and specifically cleaves uridine-rich sequences, making it
the ideal partner to cleave the RNA sequence to which TTP binds (56-58). We show that
this chimeric protein binds metal ions and can selectively bind to and catalytically cleave
the AU-rich target sequence of TTP.
6.2 Materials and Methods
6.2.1 Design of TTP2D-RNase4/RNase4 Constructs
TTP2D DNA, corresponding to amino acids 93 – 164 of murine Tristetraprolin, was
cloned from a previously constructed TTP2DpET15b vector. The forward primer was
designed to engineer an NdeI restriction site at the 5’ end of the TTP2D DNA sequence.
The reverse primer added an additional DNA fragment corresponding to (Gly4Ser)3 to the
C-terminal end of the TTP2D DNA as well as a BamHI restriction site (40). The
amplified segment was inserted into a pET22b vector using NdeI and BamHI restriction
enzymes. DNA corresponding to full length human RNase4 inserted into a pCMV-
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SPORT6 vector was purchased from transOMIC (Huntsville, AL). The RNase4 DNA
was amplified and flanked by BamHI and NotI restriction sites. This segment was
digested and added to the 3’ end of the TTP2D(Gly4Ser)3 pET22b construct. The addition
of a stop codon at the 3’ end of the RNase4 sequence prohibited the expression of the C
terminal hexahistidine tag present in the pET22b vector. The sequence of this TTP2DRNase4 was confirmed by DNA sequencing at the Biopolymer/Genomic Core Facility
housed at the University of Maryland School of Medicine. The final protein sequence is
SRYKTELCRTYSESGRCRYGAKCQFAHGLGELRQANRHPKYKTELCHKFYLQG
RCPYGSRCHFIHNPTEDLALGGGGSGGGGSGGGGSQDGMYQRFLRQHVHPEET
GGSDRYCTLMMQRRKMTLYHCKRFNTFIHEDIWNIRSICSTTNIQCKNGKMNCH
EGVVKVTDCRDTGSSRAPNCRYRAIASTRRVVIACEGNPQVPVHFDG, with the
linker sequence between TTP2D and RNase4 underlined. The RNase4 protein alone was
cloned from the purchased RNase4pCMV-SPORT6 vector and flanked by NdeI and
BamHI restriction sites prior to insertion into a pET22b expression vector. A mutant
construct in which cysteine 147 of TTP was mutated to arginine was prepared using DNA
purchased from transOMIC that corresponds to TTP2D(C147R)(Gly4Ser)3 flanked by
NdeI and BamHI sites. This DNA as well as the DNA for wild type TTP2DRNase4pET22b was digested using NdeI and BamHI and separated by gel
electrophoresis.
The
bands
corresponding
to
the
DNA
encoding
for
TTP2D(C147R)(Gly4Ser) 3 and RNase4pET22b were gel purified and ligated together to
create TTP2D(C147R)-RNase4 construct. Another mutant construct was prepared in
which histidine 12 of RNase4 was mutated to alanine (TTP2D-RNase4(H12A)) using a
Quikchange Mutagenesis Kit (Agilent)
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6.2.2 Expression and Purification of TTP2D-RNase4/RNase4 Constructs
All TTP2D-RNase4/RNase4 constructs were expressed and purified in a similar manner.
The pET22b vector containing the gene of interest was transformed into BL21(DE3)
Escherichia coli cells (Novagen). Cells were grown overnight at 37°C with Luria-Bertani
(LB) broth containing 100 µg/mL ampicillin. Overnight cultures were used to inoculate
1L of LB broth containing 100 µg/mL ampicillin. Cells were grown to mid-log phase at
30°C and expression was induced with 100 µM isopropyl β-D-1-thiogalactopyranoside
(IPTG; Research Products International Corp). Cells were grown for 4 hours post
induction and harvested by centrifugation at 7800g and 4°C for 15 minutes. A 1L cell
pellet was resuspended in 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0,
10 mM dithiothritol (DTT) and 8 M Urea to remove the protein from inclusion bodies.
The resuspension solution contained a mini ethylenediaminetetraacetic acid (EDTA) free
protease inhibitor tablet (Roche). Cells were then lysed via sonication on a Sonic
Dismembrator Model 100 (Fisher). Cell debris was removed by centrifugation for 15
minutes at 12,100g and 4°C. The bacterial supernatant was loaded onto an SP Sepharose
Fast Flow column (Sigma) and eluted using a stepwise salt gradient from 0 – 1 M sodium
chloride in 20 mM MES, pH 6.0, 10 mM DTT, and 4 M Urea. The cysteine thiols were
reduced with 10 mM tris(2-carboxyethyl)phosphine (TCEP) (Thermo) for 30 minutes at
room temperature. After filtration, the protein solution was further purified on a Waters
High Performance Liquid Chromatography (HPLC) 626 LC system with a Waters
Symmetry Prep 300 C18 7 μm reverse phase column. An acetonitrile gradient containing
0.1 % trifluoroacetic acid (TFA) was used and all proteins eluted at 31% acetonitrile with
TFA. After elution, proteins were lyophilized in a Thermo Savant SpeedVac concentrator
162
housed in a Coy anaerobic chamber (97% N2/3% H2). After lyophilization, the proteins
were resuspended in approximately 4 – 5 mL of 10 mM MES, pH 6.0 and 2 M Urea. The
protein was then quantitated on a Lamda 25 UV-visible Spectrometer using BeerLambert’s law and extinction coefficients (ε) determined by Protein Calculator v3.4
developed by Scripps Research Institute (ε278 = 12,100 for TTP2D-RNase4; ε278 =
11,400 for RNase4). To ensure adequate metal binding, 10 molar equivalents of 100 µM
zinc chloride (Fisher) was added to the unfolded protein. The protein solution was then
added to 50 mL of the redox/refolding buffer (0.1 M Tris, pH 8.0, 2 M Urea, 0.5 M LArginine, 2 mM reduced glutathione, 0.5 mM oxidized glutathione) and incubated in the
anaerobic chamber for one hour before being moved to 4°C overnight. The following
day, the protein was concentrated to 1 mL using a Pierce Concentrator 9K MWCO
(Thermo) before being buffer exchanged into 50 mM MES, pH 6.0, 50 mM NaCl, and
0.5 M L-Arginine. The buffer exchanged protein, typically 2 – 4 mL, was aliquoted and
stored at -80°C until use.
6.2.3 Metal Binding Analysis: UV-visible Spectroscopy
After lyophilization, TTP2D-RNase4 was resuspended in water and the concentration
was determined as described above. The pH was then slowly brought up to 6 in the
presence of two molar equivalents of CoCl2 by the gradual addition of 50 mM MES, 50
mM NaCl, and 40 mM glycine in a final volume of 1 mL. Binding of Co(II) to TTP2DRNase4 was assessed on the Lamda 25 UV-visible Spectrometer.
163
6.2.4 Metal Binding: Inductively Coupled Plasma Mass spectrometry (ICP-MS)
500 nM – 1 µM protein was prepared in a final volume of 15 mL in 2% trace metal grade
nitric acid (Fisher). Each sample contained an internal standard of 250 ppb scandium, 25
ppb germanium and 25 ppb indium (VHG labs) for accurate quantitation. Metal content
of protein samples as well as a blank sample (2% trace metal grade nitric acid with
standards) was measured on an Agilent 7700x ICP-MS instrument. The Zn(II) content in
the blank was subtracted from the Zn(II) content in the samples. The values were then
corrected for drift by multiplying by the drift correction factor. This correction factor is
the ratio of the expected value (in the blank) to the value in the sample of scandium,
germanium, and indium. The three drift correction factors are averaged and the Zn(II)
value is multiplied by this factor to obtain an accurate metal reading.
6.2.5 Circular Dichroism(CD)
Far-UV CD was performed on a Jasco-810 Spectropolarimeter. 15 – 20 µM protein was
buffer exchanged into 25 mM Phosphate pH 6.0 using Ultracel 3K Membrane
Centrifugal Filter Units (Millipore).
The protein was re-quantified and 300 µL was
added to a 1 mm path length quartz rectangular cell (Starna Cells). CD spectra were
collected from 190 nm to 260 nm, with a scan rate of 100 nm/min, at 25°C. A total of 5
scans were obtained per run and the average was displayed. All proteins were analyzed at
least three times.
164
6.2.6 Analysis of TTP2D-RNase4 activity
The RNA cleavage activity of TTP2D-RNase4 was measured using a
Fluorescence Resonance Energy Transfer (FRET)-based assay.
RNA substrates
incorporating a canonical TTP-binding site (ARE16, 5’-UUAUUUAUUUAUUUAG-3’)
(35) or a polyuridine RNase4 cleavage site (59) not expected to bind the TTP2D-RNase4
(R4C, 5’-CACAGAUUUUGAACAG-3’) conjugated to 5’-cyanine 3 (Cy3) and 3’fluorescein (Fl) dyes were purchased from Sigma. For intact RNA substrates, emission
from the 3’-Fl is suppressed in solution by loss of energy to the 5’-Cy3 via FRET (60).
However, since a single cleavage event within each RNA uncouples its associated dye
pair, RNA digestion can be monitored by the resulting increase in fluorescein emission.
RNA digestion reactions were assembled with 500 nM RNA and 100 pmol TTP2DRNase4 in 10 mM Tris·HCl [pH 8] containing 100 mM KCl, 2 mM MgCl2, 2 mM DTT,
and 5 µM ZnCl2. Reaction progress was monitored by measuring fluorescein emission
(λex = 485 nm, λem = 520 nm, 10 nm bandpass) every 10 seconds using a Cary Eclipse
spectrofluorometer.
Reaction progress (pmol RNA hydrolyzed) was calculated by
comparing fluorescein emission at each time point to that of a completely digested
substrate (i.e., where FRET efficiency = 0). Initial reaction velocity (V0) was calculated
from the linear phase of each time course, generally within the first minute.
165
6.3 Results and Discussion
6.3.1 Design and Construction of TTP2D-RNase4
The strategy for the design of TTP2D-RNase4 was based upon the successes reported
for ZFNs, which are chimeric constructs composed of an N-terminal ZF domain linked to
a C-terminal nuclease domain. TTP2D-RNase4 was constructed in the same manner with
TTP2D at the N-terminus of the chimeric protein and RNase4 at the C-terminus. The two
domains are liked with a (Gly4Ser)3 linker sequence, which is commonly used in the
creation of ZFN because it creates a flexible interface between the two proteins in the
chimera such that one does not interfere with the function of the other (61). The
molecular biology strategy involved cloning and inserting the DNA that encodes for
TTP2D(Gly4Ser)3 into a pET22b vector using NdeI and BamHI restriction sites, followed
by the insertion of the DNA corresponding to RNAse4 at the 3’ end of the
TTP2D(Gly4Ser)3 DNA with BamHI and NotI restriction sites. The final DNA construct
encoded for the TTP2D(Gly4Ser)3RNase4 protein, termed TTP2D-RNase4 (Figure 6.4).
The DNA sequence of this chimeric construct was verified at the University of Maryland
Biopolymer/Genomic Core Facility. As a control, the gene encoding for RNAse4 was
also cloned into a pET22b expression vector.
Additional constructs of the TTP2D-RNase4 protein were prepared as controls to
assess RNA binding and cleavage. The first construct has cysteine 147 of TTP2D
mutated to an arginine residue [TTP2D(C147R)-RNase4]. This mutation disrupts the
interaction of TTP with RNA and the chimera is expected to show slower rates of RNA
cleavage because it lacks the ARE binding specificity of TTP2D (33). The second
166
construct has histidine 12 of RNase4 mutated to an alanine residue. This is expected to
abolish catalytic activity based upon data for a homolog of RNase4, RNaseA (62). The
lack of RNase4 activity will allow us to utilize the TTP2D-RNase4(H12A) to confirm
that TTP2D binds RNA with the predicted nanomolar affinity (32, 35, 36, 40, 63, 64).
6.3.2 Expression and Purification of TTP2D-RNase4/RNase4
TTP2D-RNase4 constructs were expressed in an E. coli expression system. Protein
synthesis was induced with 100 µM IPTG at 30°C. Like TTP2D, these constructs were
(a)
25 kDa
TTP2D-RNase4
(23 kDa)
20 kDa
(b)
123 45 6 7
8
Figure 6.5 Purification of TTP2D-RNase4. (a) SDS-PAGE of purification. Lanes
1 – 7 are from SP Sepharose purification. Lane 1: ladder; Lane 2: bacterial
supernatant; Lane 3: flow through; Lane 4: wash 1; Lane 5: wash 2 – 100 mM
NaCl; Lane 6: wash 3 – 600 mM NaCl; Lane 7: wash 4 – 1 M NaCl; Lane 8: HPLC
purified proteins. (b) HPLC chromatogram of wash 3 (lane 6) from SP Sepharose
column. The black arrow indicates the collected protein peak.
167
isolated in inclusion bodies. In order to remove the proteins from inclusion bodies, each
protein was purified under denaturing conditions in the presence of urea via an SP
Sepharose column followed by reduction of the protein with TECP to reduce all cysteine
residues. The reduced protein is then further purified via HPLC and subsequently
lyophilized (Figure 6.5). As RNase4 has four disulfide bonds in its native form, it needs
to be carefully refolded in a “redox/refolding” buffer, which contains both reduced and
oxidized glutathione (57, 59, 65). Refolding in the redox/refolding buffer was performed
after lyophilization in the presence of zinc chloride such that the TTP2D portion could
fold around the zinc ion. The folded protein was subsequently buffer exchanged into 50
mM MES (pH 6.0) and 50 mM NaCl with 0.5 M L-arginine used as a solubilizing agent.
6.3.3 Co(II) Binding of TTP2D-RNase4
In order to determine if TTP2D-RNase4 binds metal ions in a similar manner to
Figure 6.6 Co(II) Binding of TTP2D-RNase4. UV-visible spectrum of apo-TTP2DRNase4 (blue) and Co(II)-TTP2D-RNase4 (red). Inset: Alignment of d-d transition of
Co(II)-TTP2D-RNase4 (red) and Co(II)-TTP-2D (purple).
168
TTP2D, cobalt was used as a spectroscopic probe. Co(II) is often used as a probe for the
spectroscopically silent Zn(II) ion in order to analyze the metal binding sites of ZF
proteins (66-68). The d7 electron count of Co(II) gives this metal rich spectroscopic
properties and upon binding of Co(II) to a ZF domain, d-d transitions appear between 550
– 750 nm, which are indicative of tetrahedral geometry (69). The shape and position of
these d-d transitions reflect the metal ligand set and the geometry at the metal center (69,
70). Any alteration of the metal binding domain will result in differences in these d-d
transitions. This technique has been extensively used to study metal binding of TTP (40,
63, 64, 67, 71).
Two molar equivalents of Co(II) were added to the apo, unfolded TTP2D-RNase4
protein and the pH was slowly raised by the addition of buffer. Analysis of the UVvisible spectrum of this complex showed the appearance of d-d transitions at 620, 650
and 685 nm. This exactly matches the maxima of the d-d transitions of TTP2D and the
spectra of the two proteins perfectly overlap (Figure 6.6) (1, 2, 40, 63, 64, 67, 71).
Therefore, the CCCH ZF domains of TTP2D-RNase4 bind Co(II) in a tetrahedral
geometry similar to TTP2D.
6.3.4 Zn(II) Stoichiometry of TTP2D-RNase4
Binding of the metal ion to the folded TTP2D-RNase4, as well as the variants, was
Table 6.1 ICP-MS Analysis of TTP2D-RNase4, RNase4, and varients. “n/d” indicates
not determined.
Protein
Equivalents of Zn(II) Post Purification
TTP2D-RNase4
2.7
RNase4
0
TTP2D(C147R)-RNase4
2.6
TTP2D-RNase4(H12A)
2.6
169
Equivalents of Zn(II) Post PD10 Column
1.5
n/d
1.3
n/d
assessed using Inductively Coupled Plasma – Mass Spectrometry (ICP-MS). TTP2DRNase4, TTP2D(C147R)-RNase4, and TTP2D-RNase4(H12A) were each isolated with
~2.5 molar equivalents of Zn(II) per protein. RNase4 by itself did not contain Zn(II) ions,
indicating the Zn(II) was binding to the TTP2D portion of the chimeric construct. Any
adventitiously bound metal was removed using a PD10 desalting column and the protein
was further analyzed via ICP-MS. At this stage, 1.3 – 1.5 molar equivalents of Zn(II)
were still bound, presumably in the CCCH ZF domains as this interaction was not labile
(Table 6.1). This analysis matches other reports for TTP metal binding. (36, 67).
6.3.5 Folding of TTPRNase4 Constructs
TTP, when bound to Zn(II), adopts a ‘disc-like’ structure which consists of a series
loops with some alpha helices (Figure 6.2a) (72). RNase4 consists of a few loops, but is
predominantly alpha helix and beta strand (Figure 6.4) (59, 65). To determine the fold of
TTP2D-RNase4, CD was performed on each variant as well as RNase4 itself. The CD
spectra of both apo and Zn(II)-TTP2D have been reported in the literature (64). This
Figure 6.7 CD Spectra of TTP2D-RNase4 Variants. Spectra of TTP2D-RNase4 (solid
black line); RNase4 (solid blue line); TTP2D(C147R)-RNase4 (solid purple line); and
TTP2D-RNase4(H12A) (dashed red line).
170
analysis shows the apo form to be largely unstructured and the addition of Zn(II) results
in an additional negative signal around 230 nm. While the CD spectra of TTP2DRNase4, TTP2D(C147R)-RNase4, and TTP2D-RNase4(H12A) look identical, they lack
the signal at 230 nm that is present in TTP2D. Instead, they more closely resemble the
CD spectra of RNase4, indicating the structure from the larger RNase4 portion of the
chimeric protein predominates in the CD spectra (Figure 6.7).
6.3.6 RNA Hydrolysis by TTP2D-RNase4
TTP interacts with the AU-rich sequence UUUAUUUAUUU with low nanomolar
affinity while RNase4 specifically cleaves U-rich sequences (32, 35, 36, 40, 56, 57, 6365). To test the ability of the chimeric TTP2D-RNase4 protein to specifically cleave the
AU-rich sequence, a FRET based assay was performed (by the Wilson laboratory)
(Figure 6.8). For these assays, the RNA sequence of interest was labeled at the 3’ end
with fluorescein (Fl) and at the 5’ end with cyanine 3 (Cy3). When the RNA is intact,
FRET is expected to occur between the two fluorophores, resulting in a decrease in the
emission of Fl. The FRET efficiency (EFRET) is inversely related to the distance between
Figure 6.8 Representation of FRET Assay. Intact RNA experiences FRET between the
donor and acceptor fluorophores. Once TTP2D-RNAse4 is introduced, the FRET efficiency
will decrease as the flourophores on the cleaved RNA separate.
171
Figure 6.9 RNA Hydrolysis by TTP2D-RNase4. Hydrolysis of the AU-rich sequence
ARE16 (closed circles) and the control R4C sequence (open circles)
the two fluorophores, thus when the distance between the donor (Fl) and acceptor (Cy3)
molecules increases, such as when RNA has been cleaved, the E FRET will decrease. This
change in EFRET can be monitored to determine the rate of RNA hydrolysis (60, 73, 74).
Performing these experiments with TTP2D-RNase4 and the AU-rich sequence, the
chimeric protein hydrolyzed the RNA at a rate of 102 ± 3 pmol min-1. The rate of
hydrolysis for a control RNA sequence that is not AU-rich was measured as 13 ± 1 pmol
min-1 (Figure 6.9). Thus, we have successfully created a novel ZF based ribonuclease that
specifically cleaves the AU-rich mRNA target sequence of TTP more rapidly than a
random sequence.
6.4 Conclusions
We have prepared the first TTP based ribonuclease, TTP2D-RNase4. Initial kinetics
studies indicate that this chimera cleaves the AU-rich target mRNA more rapidly than it
cleaves a random RNA oligomer sequence. Our strategy has involved creating a chimeric
protein composed of the RNA binding ZF domains of TTP and the RNA cleaving protein
172
RNase4. Future studies will involve (i) investigating the rates of cleavage of
TTP2D(C147R)-RNase4 to probe sequence specificity and (ii) measuring RNA binding
with TTP2D-RNase4(H12A) to determine binding affinities.
If these experiments
indicate that this first generation TTP2D-RNase4 is viable, the cytotoxicity of TTP2DRNase4 as well as its ability to cleave RNA in a cell model will be pursued.
Alternatively, modifications to the chimera (e.g. linker length, linker sequence,
ribonuclease used, and the order TTP/RNase in the chimera) will be made should this
first generation construct exhibit sub-optimal properties. Once a satisfactory TTP2DRNase prototype has been obtained and found to be active in vitro and in cells, methods
to deliver TTP2D-RNase4 to target cells will be pursued (e.g. liposomes). Overall, the
long term goal of this project is to develop a new method to target inflammation and/or
cancer based upon the TTP ZF framework.
173
6.5 References
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human tristetraprolin tandem zinc finger peptide with AU-rich element-containing
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sequence elements required for high affinity binding by the zinc finger domain of
tristetraprolin: conformational changes coupled to the bipartite nature of Au-rich
MRNA-destabilizing motifs, J. Biol. Chem. 279, 27870-27877.
36.
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Wilson, G. M. (2006) Substrate dependence of conformational changes in the
RNA-binding domain of tristetraprolin assessed by fluorescence spectroscopy of
tryptophan mutants, Biochemistry 45, 13807-13817.
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184
Chapter 7
Conclusions and Future Directions
7.1 Zinc Finger Proteins
Zinc finger (ZF) proteins were first discovered in 1985 in studies of the Xenopus
laevis protein transcription factor III A (1). When isolated, this multi domain protein was
associated with zinc ions, which were found to be essential for DNA binding (1, 2). It
was later determined that four conserved residues in each domain (CysCysHisHis) serve
as ligands to coordinate the zinc ions in a tetrahedral geometry. Thus, classical ZF
proteins were discovered. Since this discovery, over 13,000 classical ZF sites have been
identified (3). Additionally, at least 13 other types of ZFs, collectively called nonclassical ZFs, have been identified. These differ from classical ZFs in sequence, metal
coordinating ligands, (i.e. number and order of cysteine and histidine residues), fold, and
function (4-7). With the continued discovery of both classical and non-classical ZFs, it is
estimated that 3 – 5% of the entire human genome encodes for these types of proteins (8,
9). Given the ubiquity of these types of domains, ZFs exert a diverse array of biological
functions including control of gene expression. Through their roles in transcriptional and
translational regulation, they are absolutely essential in the development, growth, and
survival of eukaryotic organisms (4, 5, 8, 10).
185
7.2 Neural Zinc Finger Factor/Myelin Transcription Factor Zinc Fingers
The Neural Zinc Finger Factor/Myelin Transcription Factor (NZF/MyT) family of ZF
proteins were discovered in the early 1990s and is composed of three proteins: Neural
Zinc Finger Factor 1 (NZF-1), Myelin Transcription Factor 1 (MyT1), and Suppression
of Tumorigenicity 18 (ST18) (11-13). Although only three members have been identified
to date, these proteins play critical roles in the development of the central nervous
system. These non-classical ZF proteins contain multiple ZF domains arranged in
clusters. Each domain contains five absolutely conserved cysteine and histidine residues
arranged in a CCHHC motif (Chapter 1) (4, 5). Some of these domains have been
implicated DNA recognition; however, the roles of most of these domains have not yet
been determined (11-14). Moreover, the mode(s) of DNA recognition remains
unresolved. The factors that determine how these proteins regulate transcription are still
being discovered and this is of increasing importance given their association with a
number of serious diseases and disorders (15-35). The major goal of my thesis research
has been to understand what factors drive the function of these proteins, i.e. to determine
the mechanism of zinc mediated DNA recognition.
7.2.1 Metal Binding of the NZF/MyT Family
Upon the discovery of MyT1 in 1992, it was presumed that the NZF/MyT family of
proteins were ZFs based on the cysteine and histidine repeats present in their amino acid
sequence (11). The presence of five possible ligands for zinc in their ZF domain
(CCHHC) was intriguing – structural zinc sites typically have four ligands that coordinate
in a closed shell, tetrahedral environment. With five ligands, it is possible that a 5
186
coordinate open shell geometry may be achieved. In the late 1990s, studies that examined
the metal binding properties of NZF-1 were reported (36). Using Co(II) as a
spectroscopic probe for Zn(II), it was determined that only four ligands can coordinate
the metal ion: three cysteines and one histidine (36). UV-visible and NMR spectroscopy
studies identified the second histidine residue as the histidine involved in metal ion
coordination and the role of the first, non-metal coordinating, histidine remained
undefined, although it was proposed to be involved in a stacking interaction to help
stabilize the fold of the protein (37).
ZF proteins are initially classified as such based solely on the cysteine and histidine
repeats present in their amino acid sequence. However, during neuronal development,
iron levels are elevated and iron is also upregulated in a variety of neurological disorders.
Moreover, there is evidence in the literature that iron can bind to sites in ZF proteins and
potentially alter the function of the protein (2, 38-42). Thus, initial studies in our
laboratory focused on the ability of iron(II) to coordinate to the zinc(II) sites of a two
domain construct of NZF-1, NZF-1-F2F3. We determined that that iron(II) can
coordinate to these CCHHC domains. Remarkably, iron(II) coordination did not alter the
ability of NZF-1 to selectively bind to its DNA partner when compared to the zinc bound
form (Chapter 2) (43). This alternate metal ion coordination is potentially a mechanism
for iron toxicity because iron, unlike zinc, is redox active (39). We have also successfully
isolated and measured metal and DNA binding to the C-terminal ZF clusters of NZF-1
and MyT1: NZF-1-F456 and MyT1-F4567 (Appendix I). This is the first time that these
constructs have been isolated and shown to bind both Co(II) and Zn(II).
187
7.2.2 Role of the Non-Metal Coordinating Histidine Residues in NZF-1
A major focus of my doctoral research has been to determine the molecular level
details of the recognition event between NZF-1-F2F3 and βRAR DNA. Towards this goal,
I sought to define the role of the first histidine within each ZF sequence. This histidine
does not participate in metal coordination, yet is absolutely conserved. From structural
data, it was proposed that this histidine is involved in a pi stacking interaction with a
nearby tyrosine residue in order to stabilize the fold of the protein. (36, 37). I sought to
test this hypothesis. My approach involved mutagenesis of the first histidine residue to a
phenylalanine residue in a two domain construct of NZF-1 (NZF-1-F2F3) to preserve the
function of NZF-1 if the histidines were involved in pi-stacking. I found that the mutants
were capable of binding either Co(II) or Zn(II); however, DNA binding was completely
abolished when zinc is bound. (Chapter 3) (44). This suggested that the role(s) of this
histidine residue is more than just promoting pi stacking. From amino acid sequence
analysis, I predicted that this histidine was involved in a hydrogen bonding interaction.
Subsequent mutagenesis studies involving metal and DNA binding supported this model.
Thus, I proposed that the first histidine is involved in a hydrogen bonding interaction that
allows this protein to fold and function (Chapter 4) (45).
The presence of the first histidine in all of the CCHHC domains suggested that it may
also play a “back up” role: if the second histidine is disrupted, the first histidine could
bind to zinc instead, allowing the protein to retain its function. I performed studies in
which the second histidine was mutated to a phenylalanine in order to determine if the
non-coordinating histidine (histidine #1) would replace histidine #2 as the metal binding
ligand and promote function. Histidine #1 did bind Zn(II) as predicted, however, the
188
resultant metal-bound protein did not interact with DNA. Together, these studies suggest
the first histidine does not bind zinc, but is functionally important for the two domain
construct of NZF-1.
7.2.3 Fold of the ZF domains of the NZF/MyT Family
When NZF-1 was discovered, it was proposed that this protein would bind zinc with a
CCHC motif, given that the spacing of these ligands was identical to the spacing of the
classical CCHH ZF ligands (12). Little was known about non-classical ZF proteins at this
time and it was proposed that NZF-1 would adopt the same ββα fold as classical ZFs
(12). The three-dimensional structure of the second ZF domain of NZF-1 was determined
in 2004 and surprisingly the structure of NZF-1 was very different to that of the classical
ZFs (37). Absent were any alpha helices or beta sheets; instead the structure involved a
series of loops. Since this time, NMR solution structures have been reported for finger 4
of MyT1 and fingers 5 and 6 of ST18 (46, 47). These structures are consistent with the
initial structure of NZF-1; they are all largely loopy in nature, and have limited helical
content. (37, 46, 48).
The limited fold of the ZF domains of NZF-1 and MyT1 makes it challenging to
assess the effects of mutations on secondary structure. Circular dichroism (CD) spectra of
these proteins exhibit features indicative of mostly random coil. This is true in both the
apo and zinc bound forms of the protein with an additional feature appearing around 220
nm in the spectra of the zinc form, indicating that some structural changes occur upon
metal binding. I have also shown that, although the non-metal coordinating histidine
residue can bind to zinc when the coordinating histidine is mutated, this alternate
189
histidine coordination likely impacts the fold of the protein as evidenced by difference in
the CD spectra (Chapter 3) (44).
7.2.4 DNA Binding by the NZF/MyT Family
Despite the importance of the NZF/MyT proteins, little is known about the
physiochemical features that drive DNA binding. These proteins were identified by
studies in which specific DNA sequences were used as “bait” to discover transcription
factors that target this DNA (11, 12). Most of the DNA sequences that have been
identified as binding targets for the NZF/MyT family contain an AAGTT motif, leading
to the proposal that this short sequence is the key recognition sequence for this family of
ZFs (11, 12, 14, 49-51). However, the presence of multiple ZF clusters in each protein
suggests that there may be more than this sequence involved in the recognition event, as
the sizes of the ZF domains is larger than the size of the short DNA target. Thus, I aimed
to identify other key features.
I have demonstrated, in vitro, that the ZF clusters of both NZF-1 and MyT1 bind to
the same DNA sequence with affinities ranging from low nanomolar to micromolar
(Chapter 3; Appendix I). My most significant finding is that the two central ZF domains
of MyT1, comprised of finger 2 and finger 3, bind to the DNA target sequence of NZF-1
only non-specifically. This is striking given that these two sequences are 92% identical,
96% similar. I then identified a single amino acid to be responsible for this difference in
DNA recognition. A completely non-conserved arginine in finger 3 of NZF-1, which is
not present in MyT1, is responsible for this drastic difference in DNA binding. This
residue exists in the variable region between the two histidine residues in the ZF domain.
190
This region is the only region in the ZF domain that is not highly conserved, leading us to
the hypothesis that it is the few non-conserved amino acids within the primary amino acid
sequences of the CCHHC ZFs that drive sequence specific DNA recognition (Chapter 3)
(44).
7.3 Catalytic ZF Proteins
Another focus of my dissertation research has been to re-design ZF domains or ZF
proteins to incorporate enzymatic activity. My motivation for these studies came from the
highly successful genome editing accomplished by ZF nucleases (ZFNs) coupled with
our emerging understanding of the structural/functional relationships of non-classical
ZFs. (52-55).
7.3.1 Consensus Peptide 1 (CP-1)
I created an artificial catalytic zinc site based upon Consensus Peptide-1 (CP-1), an
optimized classical ZF, in collaboration with the laboratory of Dr. David Goldberg (JHU)
(56). I mutated the second cysteine residue in the CCHH motif of CP-1 to an alanine,
creating a CAHH domain. This single mutation created an open coordination sphere that
is essential for the activity of a catalytic zinc site (57, 58). The resultant CP-1(CAHH)
peptide bound metal via three amino acid ligands (Cys2His) and could also coordinate
two water molecules. This modified CP-1 was found to hydrolyze 4-nitrophenylacetate.
Thus, I successfully engineered an artificial catalytic zinc site using the classical ZF
domain as our base (Chapter 5).
191
7.3.2 TTP2D-RNase4
Our second protein engineering approach involved modifying Tristetraprolin (TTP) to
have ribonuclease activity (in collaboration with Dr. Gerald Wilson’s laboratory, UMB
SOM). The non-classical CCCH type ZF protein TTP is an RNA binding protein that is
essential in controlling the inflammatory response and also tumorous properties (4, 5, 5961). TTP functions by binding to the 3’ untranslated region of AU-rich mRNA, marking
the mRNA for degradation. These mRNAs can encode for cytokines and tumorpromoting proteins, thus TTP activity is essential for decreasing inflammation and has
anti-cancer effects (4, 5, 59, 61).
My approach to create TTP with ribonuclease activity was to conjugate TTP to
ribonuclease4 (RNase4), a protein which cleaves U-rich sequences (62, 63). This strategy
has strong precedence in ZFNs which are classical ZFs with nuclease activity (53-55).
TTP2D-RNase4 binds cobalt and zinc in similar manner to TTP. When zinc is bound,
TTP2D-RNase4 cleaves the AU-rich target sequence of TTP (Chapter 6).
7.4 Future Directions for the NZF/MyT Family
I have successfully cloned and purified a number of constructs of both NZF-1 and
MyT1 (Chapter 3, Appendix I). I have examined their metal binding, fold, and DNA
binding properties. Despite these advances, there remains much to be discovered about
the function of these proteins. A key questions is why this family of proteins contains
multiple ZF domains when just two domains are required for high affinity DNA binding?
My in vitro studies of different ZF clusters of NZF-1 and MyT1 with βRAR have revealed
that ZF2+ZF3 of NZF-1 binds to this target DNA with the highest affinity, thus I propose
192
that the other clusters may interact with different DNA sequences or potentially with
other proteins. In addition, sequence is often not the only important feature for a
protein/DNA binding interaction; the shape of the protein and DNA can contribute to
these interactions (64, 65). NMR and modeling studies of a construct of ZFs 4+5 of
MyT1 interacting with the β-retinoic acid response element (βRARE), which have
suggested that the overall shape of these ZF domains is important for DNA binding by
allowing the ZF domain to fit into the major groove support a role for shape in
recognition. While the shape of ZF domain may play an important role in the interaction
with DNA, our studies show that the amino acid sequence of the NZF/MyT ZF domains
are also essential in dictating DNA binding (44, 48). Additional structural studies are
needed to better understand how NZF-1 interacts with βRAR and to tease out the
contribution of sequence and shape.
A second future direction for this project is to determine how our in vitro results
translate in a cell model. We have initiated cell based studies of NZF-1 with βRAR using
a luciferase reporter system (66). My approach involves transforming a FLAG-tagged
vector of NZF-1 (full length and specific mutants) along with a luciferase reporter vector,
which contains the βRAR promoter upstream of a firefly reporter [from Dr. Cécile
Rochette-Egly (Institut Génétique Biologie Moléculaire Cellulaire)] into HEK293 cells.
By monitoring transcriptional activation as a function of protein (e.g. wild type full
length or ZF mutants) I can identify the domains that are required for transcriptional
activation. I can also test our hypothesis, which is based on in vitro data, that NZF-1F2F3 is the key binding partner for βRAR.
193
Another key question is are there additional roles for other ZF clusters? I hypothesize
that the other clusters are also involved in DNA binding. If the cell studies show that all
clusters are required for transcriptional activation, the various clusters may be interacting
with different DNA sequences in the βRAR promoter. One approach to test this
hypothesis is a SELEX type approach that probes the interaction of the segments of the
βRAR promoter with the various domains of NZF-1 (67). The various NZF-1 constructs
can also be used in Electrophoretic Mobility Shift Assay (EMSA) experiments, as
described in the conclusions of Appendix I, in which various fragments of the promoter
are tested for their ability to bind to the different clusters NZF-1 (68). These studies will
help determine which clusters of NZF-1 are binding to which sequences within the βRAR
promoter.
If only some of the ZF domains are needed for transcriptional activation, the other
clusters may interact with the promoters of different genes or with different proteins. To
determine if this is the case, ChIP-SEQ can be performed in neuronal cell lines in order to
pull out different target sequences of NZF-1 (69). In addition, SELEX with a random
pool of oligonucleotides can be performed and those DNAs that bind to NZF-1 can be
sequenced and the human genome can be parsed for potential interaction partners. To
determine any protein partners for NZF-1, pull down assays can be performed (70).
To fully understand this family as a whole, the above proposed studies can also be
performed with both MyT1 and ST18. These studies are necessary to fully understand
the role that this family of proteins plays in the body and how they are able to function.
This will not only provide greater insight into how the nervous system develops, but will
also provide a basis for understanding the function of these proteins in disease states.
194
7.5 Future Directions for Catalytic ZFs
7.5.1 CP-1
I have successfully created a novel catalytic zinc site out of an optimal classical ZF
domain. To optimize this catalytic center, the amino acid residues in the zinc domain can
be mutated in order to determine which sequence promotes optimal catalytic activity.
Once optimized, studies can be done to determine if this peptide can hydrolyze
oligonucleotides (71). If proven successful, CP-1(CAHH) may represent a novel cleavage
agent that can be engineered with a ZF domain for genome editing.
7.5.2 TTP2D-RNase4
We have successfully imparted catalytic activity to a TTP2D construct by linking it to
RNase4. We have shown in vitro that this chimera selectively cleaves ARE in mRNA and
now we seek to demonstrate this in cellulo. Using reporter plasmids, we will examine the
ability of TTP2D-RNase4 to cleave RNA in cells as well as perform toxicity studies. If
these studies prove successful, analysis of TTP2D-RNase4 activity in a cellular model of
inflammation in which tumor necrosis factor α expression is enhanced will be evaluated.
At this point, if the first generation TTP2D-RNase4 does not exhibit optimal properties,
alterations to the linker sequence, ribonuclease domain or the arrangement of the domains
in the chimera can be made. As the overall goal of this project to engineer a novel
treatment for inflammation and cancer, methods to deliver this protein will be explored
upon successful completion of these studies.
195
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Nomura, A., and Sugiura, Y. (2004) Sequence-selective and hydrolytic cleavage
of DNA by zinc finger mutants, J. Am. Chem. Soc. 126, 15374-15375.
205
Appendix I
Characterization of the C-terminal Zinc Finger Clusters of the Neural Zinc
Finger Factor family: metal ion coordination and DNA binding
I.1 Introduction
Zinc Finger (ZF) proteins are zinc co-factored metalloproteins that represent over half
of all eukaryotic transcription factors and have also been shown to interact with both
RNA and proteins (1-4). Zinc is coordinated to these sites in a tetrahedral geometry using
a combination of four cysteine and/or histidine residues (3-7). Upon zinc coordination,
the domain adopts secondary structure, which allows the protein to function by
interacting with its binding partner (5, 6). This type of domain was first documented in
the Xenopus laevis protein transcription factor III A (TFIIIA), which contains nine
‘classical’ ZF sites (8, 9). Classical ZF domains are characterized by a CCHH zinc
coordinating ligand set and a ββα fold, which the domains adopts once bound to zinc (35). Since the discovery of TFIIIA over 13,000 classical ZF domains have been discovered
(10). In addition to these domains, there are 13 other classes of ZFs, which are
collectively referred to as the ‘non-classical’ ZF proteins. These classes vary based on the
exact composition of the metal coordinating residues, the spacing between these residues,
and the fold of the metal-bound protein (3-6).
The classical ZF domains are the best studied ZF sites. These proteins are
predominately DNA binding proteins, but may also interact with RNA or other proteins
206
(a)
(b)
(c)
Figure I.1 Sequence Logo of ZF Domains. (a) Sequence logo of classical CCHH ZF
domains, generated from 13,439 sequences. (b) Sequence logo of non-classical CCCH ZF
domains generated from 1,013 sequences. (c) Sequence logo of non-classical CCHC ZF
domains generated from 679 sequences.
(11). Their interaction with DNA has been studied in such depth that the amino acid
sequence of these domains can be altered such that the protein will bind any DNA target
of interest. Thus, these ZF domains are used in the creation of ZF nucleases (ZFN), which
are being explored as a therapeutic for genetic disorders (12-14). The consensus amino
acid sequence for the classical ZF domains is CX2-5CX12-13HX3-5H, where X is any
amino acid (Figure I.1a) (15, 16). Analysis of all known classical ZF sites reveals that
there are few highly conserved amino acids outside of the metal coordinating residues.
The few amino acids that are highly conserved have been implicated in being essential
for the conserved ββα fold of the protein (16). The large degree of variability in these
207
domains is what allows for these ubiquitous transcription factors to bind a wide variety of
DNA target sequences. Specifically, four positions along the alpha helical domain of the
CCHH ZF have been shown to be responsible for sequence specific DNA recognition
(17-19).
Non-classical ZF proteins have also been shown to bind to DNA, RNA, or other
proteins; however, the specificity of these types of interactions has not been studied as in
depth as the classical ZFs (3, 4, 20, 21). As non-classical ZF domains are so diverse, no
general rules exist for the ZF/DNA (or RNA) protein interactions. Each of the 13
different classes is unique and proteins within the same class can differ significantly. For
example, the CCCH type ZF protein tristetraprolin (TTP) specifically binds to AU-rich
(a)
(b)
Figure I.2 Sequence of CCHC type ZF domains. (a) Schematic of ZF domain
arrangement of NZF-1 and MyT1. (b) Alignment of ZF domains of NZF-1 (black) and
MyT1 (blue).
208
(b)
(a)
Figure I.3 NMR Solution Structures of NZF-1 and MyT1. (a) Structure of F2 of
NZF-1 with metal coordinating residues shown in black. (b) Structure of F4 of
MyT1 with metal coordinating residues shown in blue.
sequences in the 3’untranslated region of cytokine mRNA, marking these mRNAs for
degradation (22, 23). Individual RNA bases are recognized by these ZF domains (24-27).
Another CCCH ZF protein, muscleblind-like (MBNL), sequence specifically recognizes
structured RNA. Specifically, MBNL binds to RNA that contains a hairpin or stem-loop
structure (28-30). The great differences in the binding activity of these two CCCH-type
ZF domains may be a result of the lack of amino acid conservation, with the exception of
the CCCH ligands, that exists within this family (Figure I.1b) (10). This lack of amino
acid conservation is also seen with other non-classical ZF sites, such as the CCHC type
ZF proteins that includes the HIV protein nucleocapsid (Figure I.1c) (10).
The CCHHC type ZFs known as the Neural Zinc Finger Factor/Myelin Transcription
Figure I.4 Sequence Logo of CCHHC ZF Domains. Logo includes all domains present
in NZF-1, MyT1, and ST18. Non-metal coordinating histidine residue boxed in red.
209
Factor Family (NZF/MyT) are a unique family of ZFs for which the amino acid
sequences between homologs is highly conserved (3, 4). This family of transcription
factors consists of three proteins: Neural Zinc Finger Factor 1 (NZF-1), Myelin
Transcription Factor 1 (MyT1), and Suppression of Turmorigenecity 18 (ST18), with
most research focusing on NZF-1 and MyT1 (31-33). Each of these proteins contains a
total of six or seven ZF domains arranged in clusters (Figure I.2a). A unique feature of
these domains is the presence of five absolutely conserved cysteine and histidine residues
arranged in a CCHHC motif (Figure I.2b) (3, 4). UV-visible and NMR spectroscopy
studies have shown that these domains coordinate zinc with a CCHC motif, with the
second histidine residue serving as the histidine ligand for metal ion coordination (Figure
I.3) (34-36). Mutagenesis studies have shown that the first histidine residue is important
for DNA binding and is involved in a hydrogen bonding network (37). This is likely true
for all of the DNA binding domains in this family as this CCHHC motif is completely
conserved in all of the domains. Strikingly, almost all of the amino acids present in the
ZF domains of the NZF/MyT family are conserved (Figure I.4) (3, 4). The overall
consensus sequence for these zinc domains is CPXPGCXGXGHX7HRX4C, with a
variable region present between the two histidine residues. This high degree of sequence
conservation has led to the hypothesis that each ZF domain in this family recognizes the
Figure I.5 Sequence Logo of DNA Target Sequences. Logo includes known and
proposed DNA target sequences. The AAGTT site is boxed.
210
same DNA target sequence, AAGTT (31, 32, 38-40). While this sequence appears
frequently in DNA targets that this family has been shown to interact with (Figure I.5),
recent studies have shown the central two ZF domain of NZF-1 and MyT1 (F2+F3) bind
to this target sequence with significantly different affinities (31, 32, 36, 38, 41-43). This
construct of NZF-1 binds specifically with a dissociation constant (Kd) in the low
nanomolar regime, while the analogous construct of MyT1 binds only non-specifically to
this target. A single amino acid in NZF-1 is responsible for this difference in DNA
binding (36). How the other ZF domains in this family of proteins interact with this DNA
target has not been explored.
Here, we report preliminary studies to determine the metal ion and DNA binding
properties of the C-terminal ZF domains of both NZF-1 and MyT1. We have isolated the
C-terminal 3 ZF cluster of NZF-1 (F4+F5+F6) and the C-terminal 4 ZF cluster of MyT1
(F4+F5+F6+F7) to examine their ability to bind cobalt and zinc. We present evidence
that these clusters bind to the DNA target sequence of the central ZF cluster of NZF-1,
which contains that AAGTT sequence, with differing binding affinities.
I.2 Materials and Methods
I.2.1
Cloning, Expression, and Purification of NZF-1-F456 and MyT1-F467
A DNA fragment corresponding to residues 895 – 1037 of full length NZF-1 from Rattus
norvegicus, termed NZF-1-F456, was amplified via the Polymerase Chain Reaction
(PCR) from a R. norvegicus brain cDNA library, which was a generous gift from Dr.
Anthony Lanahan (Yale University). This DNA fragment was subsequently ligated into a
211
pGEX2T vector with an N-terminal glutathione-s-transferase (GST) tag and a TEV
protease linker site. The DNA corresponding to residues 592 – 777 of full length MyT1,
termed MyT1-F4567 was cloned in an analogous manner. For expression, the vectors
were transformed into BL21(DE3) Escherichia coli (Novagen) cells and the cells were
then grown in Luria Bertani (LB) broth with 100 µg/mL ampicillin. For NZF-1-F456, the
cells were grown at 37°C until mid-log phase. Protein expression was induced with 1 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were grown for 2 hours post
induction. For MyT1-F4567, the cells were grown at 37°C until they reached an OD600 of
0.4. At this point, the temperature was decreased to 18°C and the cells were grown until
an OD600 between 0.6 – 0.8. Protein expression was induced with 50 µM IPTG and
grown for 24 hours post induction. Cells were harvested by centrifugation at 7800 x g for
15
minutes
at
4°C.
Cell
pellets
were
resuspended
in
50
mM
Tris
[tris(hydroxymethyl)aminomethane] at pH 7.5, 150 mM NaCl, 1% Triton X-100, and 5
mM dithiothreitol (DTT) per ZF. Each cell suspension also contained a mini
ethylenediaminetetraacetic acid (EDTA) free protease inhibitor tablet (Roche). Cells were
lysed via sonication on a Misonix Sonicator 3000. Cell debris was removed by
centrifugation at 12100 x g for 15 minutes at 4°C. The bacterial supernatant was loaded
onto a glutathione agarose column (Sigma) and purified following the manufacturer’s
protocol. The GST tag was removed from the protein by incubation with 50 units of
ProTEV Protease overnight at room temperature. The following morning, the cysteine
thiols
of
the
protein
were
reduced
by
incubation
with
10
mM
tris(2-
carboxyethyl)phosphine (TCEP) (Thermo) at room temperature for 30 minutes. The
protein was further purified via High Performance Liquid Chromatography (HPLC) using
212
a Waters 626 LC system and a Waters Symmetry Prep 300 C18 7 µm reverse phase
column with an acetonitrile gradient containing 0.1% trifluoroacetic acid (TFA) before
being dried using a Thermo Savant SpeedVac concentrator housed in a Coy anaerobic
chamber (97% N2/3% H2).
I.2.2
Metal Binding Studies
Metal titrations were performed on a PerkinElmer Lambda 25 UV-visible Spectrometer.
15 - 25 µM apo-peptide was titrated with various molar equivalents of CoCl2 and the
absorbance was monitored until saturation. ZnCl2 was then titrated into the Co(II)-ZF at
equal molar equivalents and the decrease in absorbance was monitored. The relative
affinity of the peptides for these metals was determined following the method developed
by Berg and Merkle (44). All titrations were performed in 200 mM HEPES [4-(2hydroxyethyl)-1-piperazineethanesulfonic acid], 100 mM NaCl at pH 7.5. Data were fit
to an appropriate binding equilibria using linear least squares analysis (KaleidaGraph,
Synergy Software).
I.2.3
Fluorescence Anisotropy (FA) Studies
FA assays were performed on an ISS PC-1 spectrofluorimeter configured in the L format
with a wavelength/band pass of 495 nm/2 nm for excitation and 517 nm/1 nm for
emission were utilized. Experiments were performed in 50 mM HEPES, 100 mM NaCl at
pH 7.5 in a Spectrosil far-UV quartz window fluorescence cuvette (Starna Cells). Binding
studies
were
performed
with
10
nM
fluorescently
labeled
β-RAR
DNA,
CACCGAAAGTTCACTC, which had been annealed with its complementary strand
213
(IDT). All titrations were performed in the presence of 0.05 mg/mL bovine serum
albumin (BSA) to prevent adherence of DNA or protein to the cuvette walls. The
anisotropy, r, was monitored as protein was added in increments to the fluorescently
labeled DNA. Each data point represents the average of 60 readings taken over a period
of 115 seconds. To normalize the data, anisotropy values were converted to fraction
bound, Fbound (fraction of DNA bound to peptide at a given DNA concentration),
according to the following equation:
Fbound 
r  r free
(rbound  r )Q  (r  r free )
where rfree is the anisotropy of fluorescently labeled DNA and rbound is the anisotropy of
the peptide-DNA complex at saturation. Q is the quantum yield that is applied as a
correction factor to account for changes in fluorescence intensity over the course of the
experiment (Q = Ibound/Ifree). Typically, Q values ranged from 0.65 – 0.75 Fbound was then
plotted against peptide concentration and the data fit to a one-site binding model:
P+D
Kd 
Fbound 
PD
[ P][ D]
[ PD ]
Ptotal  Dtotal  K d  ( Ptotal  Dtotal  K d ) 2  4 Ptotal Dtotal
2 Dtotal
where P is the protein concentration and D is the DNA concentration.
214
I.3 Results and Discussion
The NZF/MyT family of ZF proteins is emerging as an increasingly important family
of transcription factors given that they are essential to development and their
miregulation is linked to a number of diseases and disorders (31, 32, 45-70). Despite this
importance, little is known about their metal and DNA binding properties. Our lab and
others have studied in depth the metal interactions of F2+F3 of both NZF-1 and MyT1
and have begun to understand the factors that contribute to the DNA recognition
properties of these fingers (35-37, 39, 71). However, less is known about the C-terminal
ZF clusters of this family. We sought to begin to understand these clusters more by
preliminarily examining the metal and DNA binding properties of NZF-1-F456 and
MyT1-4567.
(a)
(b)
(1) (2) (3) (4)
(5) (6) (7) (8)
Figure I.6 Expression of NZF-1-F456 and MyT1 F4567 (a) Expression of NZF-1-F456.
Lane 1: ladder; Lane 2: pre-induction; Lane 3: 1 hr post-induction (1 mM IPTG); Lane 4: 2
hr post-induction. The GST bound protein, 41.4 kDa, is boxed. (b) Expression of MyT1F4567. Lane 5: ladder; Lane 6: pre-induction; Lane 7: 6 hr post induction (50 µM IPTG);
Lane 8: 24 hr post-induction. The GST bound protein, 45.6 kDA, is boxed.
215
(a)
(b)
Figure I.7 Purification of NZF-1-F456 and MyT1-4567. (a) GSH agarose purification of
GST bound NZF-1-F456 (41.4 kDa). The cleavage reaction results in the separation of GST
(26 kDa) from NZF-1-F456 (15.4 kDa). (b) GSH agarose purification of GST bound MyT14567 (45.6 kDa). The cleavage reaction results in the separation of GST (26 kDa) from MyT1F4567 (19.6 kDa).
I.3.1
Expression and Purification of NZF-1-F456 and MyT1-4567
The constructs of NZF-1 and MyT1 that have been extensively studied in our lab are
expressed and purified without the use of affinity tags (36, 37, 71). NZF-1-F456 and
MyT1-F4567 were engineered with an N-terminal GST tag to more easily immobilize
these protein constructs on a column for future studies. While NZF-1-F456 expressed in a
similar manner as NZF-1-F23 and MyT1-F2F3, MyT1-4567 showed no expression under
these conditions, which include E. coli growth at 37°C and induction of protein synthesis
with 1 mM IPTG (36, 37, 71). After many expression trials in a variety of conditions,
successfull MyT1-F4567 expression was seen at 18°C with 50 µM IPTG (Figure I.6).
Both protein constructs expressed at much lower levels than the untagged F2F3
constructs.
Purification of the GST constructs and the cleavage of the tag from the ZF domains
were performed according to the manufacturer’s protocols. A variety of conditions were
216
attempted to obtain full cleavage of the GST tag from the ZF domains of NZF-1 and
MyT1 including altering the length of time the protein underwent cleavage as well as the
temperature of the cleavage reaction (room temperature versus 30°C). The highest degree
of cleavage was seen after 24 hours and the change in temperature did not appear to alter
the degree of cleavage. Almost all of the GST tag was successfully cleaved from NZF-1F456. Full cleavage of the GST tag was never obtained for MyT1-F4567; however, some
untagged MyT1-4567 was isolated (Figure I.7). The three constructs (GST-ZF, GST, and
ZF) were successfully separated from each other via HPLC.
I.3.2
Metal Binding Analysis
To investigate the ability of NZF-1-F456 and MyT1-4567 to bind Zn(II), Co(II) was
(a)
(b)
(c)
Figure I.8 Co(II) and Zn(II) Titration of NZF-1-F456. (a) Schematic representation of
experimental design. The apo ZF is first saturated with Co(II) before the addition of Zn(II)
results in the replacement of this Co(II). (b) d-d transition bands that appear upon addition of
Co(II) to apo-NZF-1-F456. (c) Plot of the absorption spectrum at 679 nm as either a function of
added Co(II) to apo-NZF-1-F456 (orange) or Zn(II) to Co(II)-NZF-1-F456 (blue). The data
were fit to appropriate binding equilibria and upper limit Kds of 153 nM and 11 nM for Co(II)
and Zn(II), respectively were obtained. The solid lines represent the non-linear least squares fit.
217
used as a spectroscopic probe. Zn(II) is spectrosopically silent due to its d10 electron
count, but Co(II) displays rich spectroscopic properties as it has a d7 electron count (7274). Furthermore, Co(II) can coordinate to ZF sites in a manner similar to Zn(II) and has
been shown to be an excellent probe for the examination of metal binding in ZF domains.
Both metals bind to these sites in a tetrahedral geometry and Co(II) in this geometry
displays d-d transitions between 550 – 750 nm (15, 75). Once Co(II) is bound to zinc
sites, Zn(II) can be titrated in to the Co(II)-ZF and the Zn(II) will displace the Co(II)
because there is no ligand field stabilization energy penalty to be paid for Zn(II) in a
tetrahedral versus octahedral geometry (44). The increase in absorbance of the d-d
transitions as a result of Co(II) binding and the subsequent decrease in absorbance as a
result of Zn(II) binding can be monitored to determine Kds for these interactions (44).
This method has been used on various constructs within the NZF/MyT family (35-37, 39,
76).
Performing this assay on NZF-1-F456 and MyT1-F4567 results in the appearance
(a)
(b)
Figure I.9 Co(II) and Zn(II) Titration of MyT1-F4567 (a) d-d transition bands that
appear upon addition of Co(II) to apo-MyT1-F4567. (c) Plot of the absorption spectrum
at 679 nm as either a function of added Co(II) to apo-MyT1-F4567 (orange) or Zn(II) to
Co(II)-MyT1-4567 (blue). The data were fit to appropriate binding equilibria and upper
limit Kds of 11 µM and 336 nM for Co(II) and Zn(II), respectively were obtained. The
solid lines represent the non-linear least squares fit.
218
Table I.1 Kds of NZF-1 and MyT1
constructs for Co(II) and Zn(II)
Construct
NZF-1-F2F3
MyT1-F2F3
NZF-1-F4F5F6
MyT1-F4F5F6F7
Kd Co(II) Kd Zn(II)
70 nM 120 pM
200 nM 360 pM
153 nM 11 nM
11 µM 336 nM
of similar d-d transition bands (Figure I.8
and I.9). These appear identical to NZF-1F23 and MyT1-F23, indicating all the ZF
clusters in this family of proteins binds metal
in a similar manner (36). These bands are
indicative of Co(II) in a tetrahedral geometry with a CCHC ligand set (7). This ligand set
is in agreement with the published NMR studies of F2 of NZF-1 and F4 of MyT1 (34,
35). The dissociation constant for Co(II) binding were in the regime typically seen for
Co(II)-ZF interactions, although the Kd for MyT1-4567 was lower than what was
observed for the other clusters within this family (3, 77). Similarly, binding in the
nanomolar regime was seen for the C-terminal clusters of NZF-1 and MyT1 compared to
the picomolar regime for the F2F3 clusters (Table I.1).
I.3.3
DNA Binding of NZF-1-F456 and MyT1-F4567 to the βRAR Promoter
A bonafide DNA target has been identified for the F2+F3 cluster of NZF-1 via DNase
footprinting studies (39). This target was first used in the discovery of NZF-1 and
footprinting studies confirmed that the central ZF cluster interacted with the AAGTT
motif (31, 39). Studies have suggested that this short motif is all that is necessary for a
tight specific interaction with DNA and various target DNAs have been identified that
contain this sequence (31, 32, 38, 40-43). Our lab has demonstrated that NZF-1-F23
binds this sequence specifically with a Kd in low nanomolar regime, but MyT1-F23,
which is 92% identical to NZF-1-F23, does not (36). Given the surprising differences in
219
Table I.2 Kd of NZF/MyT
constructs for βRAR.
Kd
Construct
NZF-1-F2F3
14 nM
MyT1-F2F3
1.3 µM
NZF-1-F4F5F6 511 nM
MyT1-F4F5F6F7 937 nM
DNA binding activity of these two ZF clusters, we
sought to examine the interaction of the C-terminal ZF
clusters with this DNA sequence.
Preliminary FA studies with NZF-1-F456 and
MyT1-F4567 show that these proteins bind to the βRAR
sequence with Kds of 511 nM and 937 nM, respectively (Figure I.10). While the Kd for
NZF-1-F456 is lower than the 14 nM reported for NZF-1-F23, the Kd for MyT1-4567 is
similar to the Kd of 1.3 µM determined for MyT1-F2F3 for this sequence (Table I.2) (36).
In addition, SPR studies using a construct composed for F4+F5 of MyT1 and this same
DNA sequence has reported a Kd of 1 µM (78). These studies suggest that NZF-1-F23
may be the only ZF cluster in this family which interacts with this segment of the βRAR
promoter. This compliments work that has shown a single arginine residue that is present
in F3 of NZF-1, but in no other domains of this family, is required to specifically bind to
this target sequence (36).
(a)
(b)
Figure I.10 FA of NZF-1-F456 and MyT1-F456. Change in anisotropy (as fraction
bound) as NZF-1-F456 (a) and MyT1-4567 (b) is titrated into the βRAR DNA.
220
Figure I.11 Percent identity of NZF-1 and MyT1. Percentages indicate how identical
the designated domains are between NZF-1 and MyT1. Orange boxes represent ZF
clusters. The full length NZF-1 and MyT1 proteins are 58% identical.
I.4 Conclusion
We have examined the metal and DNA binding properties of the C-terminal ZF
cluster of NZF-1, F456, and MyT1, F4567. We have shown that these domains bind
Co(II) and Zn(II) in a manner similar to other ZF domains in this family and that they
bind to the βRAR target DNA sequence with affinities well below that of NZF-1-F23.
Although these studies are preliminary, they demonstrate a difference in DNA
binding affinity for the AAGTT target that was proposed to be all that was necessary for
the interaction with DNA. Given the previous results of studies that focused on the DNA
binding of the central two finger cluster, it is likely that the differences in affinity
observed in these studies are due to the few non-conserved amino acids present in the ZF
domain (36). The arginine residue identified as being essential for the interaction of NZF1-F23 with DNA is not present in either of the domains tested here, potentially
accounting for the decreased binding affinity (36).
Although the NZF/MyT family of ZF proteins is incredibly unique because of their
high degree of sequence conservation in their ZF domains, this conservation is only seen
within the zinc sites (Figure 11). In fact, full length NZF-1 and MyT1 are only 58%
identical. Interestingly, studies have shown that this family can participate in protein221
protein interactions and some of these interactions have been documented to take place in
the central domain between the two main ZF clusters (79, 80). NZF-1 and MyT1 only
share 45% sequence identity in this span of amino acids. This suggests that perhaps this
family may interact with different proteins in vivo. These potential protein-protein
interactions may influence the DNA binding activity of these important ZF proteins
beyond what has been documented here. It is also possible that each ZF cluster in the
NZF/MyT family recognizes a different DNA sequence in the promoters of which these
proteins bind. We have begun preliminary studies to examine this hypothesis using
MyT1. In these studies, we have obtained 10 different portions of the PLP promoter, each
72 basepairs in length and have begun electrophoretic mobility shift assays (EMSAs) to
determine which segment the two ZF and four ZF clusters of MyT1 interacts with. Any
interactions that are identified from these studies can be subjected to DNase Footprinting
to determine the exact bases that each ZF cluster recognizes. Additionally, it is not clear
how the observed differences in binding affinities in vitro impact transcriptional
activation. Studies are currently underway in our laboratory to explore this latter point
using the full length NZF-1 protein. These studies will provide greater insight into how
this essential family of ZF transcription factors interacts with their DNA targets.
222
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Krizek, B. A., Amann, B. T., Kilfoil, V. J., Merkle, D. L., and Berg, J. M. (1991)
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4518-4523.
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Michael, S. F., Kilfoil, V. J., Schmidt, M. H., Amann, B. T., and Berg, J. M.
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