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Principles of Bioinorganic Chemistry - 2003
Lecture
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Date
9/4 (Th)
9/ 9 (Tu)
9/11 (Th)
9/ 16 (Tu)
9/18 (Th)
9/23 (Tu)
9/25 (Th)
9/30 (MU)
10/2 (MU)
10/7 (Tu)
10/9 (Th)
10/16 (Th)
10/21 (Tu)
10/23 (Th)
Lecture Topic
Reading
n+
Intro; Choice, Uptake, Assembly of M Ions
Ch. 5
Metalloregulation of Gene Expression
Ch. 6
Metallochaperones; Metal Folding, X-linking
Ch. 7
Zinc Fingers; Metal Folding; Cisplatin
Ch. 8
Electron Transfer; Fundamentals
Ch. 9
Long-Distance Electron Transfer
Ch. 9
Hydrolytic Enzymes, Zinc, Ni, Co
Ch. 10
Model Complexes for Metallohydrolases
Ch. 10
Dioxygen Carriers: Hb, Mb, Hc, Hr
Ch. 11
O2 Activation, Hydroxylation: MMO, P-450, R2 Ch. 11
Model Chemistry for O 2 Carriers/Activators
Ch. 11
Complex Systems: cyt. oxidase; nitrogenase
Ch. 12
Metalloneurochemistry/MedicinalInorg. Chem. Ch. 12
Term Examination
Ch. 12
Problems
Ch. 1
Ch. 2
Ch. 3
Ch. 4
Ch. 5
Ch. 6
Ch. 7
Ch. 8
Ch. 9
Ch. 10
Ch. 11
Ch. 12
Metallochaperones; Metal Folding
PRINCIPLES:
•Metallochaperones guide and protect metals to natural sites
•Chaperone and target receptor protein structurally homologou
•Metal-mediated protein structure changes affect transcription
•Metal-mediated protein structure changes affect translation
•Metal-induced protein structure changes also activate enzymes
•Metal-induced bending of DNA affects function
•Metal ionic radii and M–L water bridging are used to advantage
ILLUSTRATIONS:
•Copper insertion into metalloenzymes
•Zinc finger proteins control transcription
•Ca2+, a second messenger and sentinel at the synapse
•Cisplatin, an anticancer drug
Zinc Fingers - Discovery, Structures
A. Klug, sequence gazing, proposed zinc fingers for TFIIIA,
which controls the transcription of 5S ribosomal RNA.
Zn2+ not removed by EDTA. 9 tandem repeats. 7-11 Zn/protein.
Y or F – X –CC – X2,4 –CC – X3 – F – X5 – L – X2H– H – X3,4
H – H – X2
The coordination of two S and 2
N atoms from Cys and His
residues was supported by
EXAFS; Zn–S, 2.3 Å; Zn–N, 2.0 Å.
Td geometry.
The protein folds only when zinc
is bound; > 1% of all genes have
zinc finger domains.
X-ray Structure of a Zinc Finger Domain
Structure of a Three Zinc-Finger Domain of Zif 268
Complexed to an Oligonucleotide Containing its
Recognition Sequence
The Specificity of Zinc for Zinc-finger Domains
Kd value:
Metal ion:
2 pM 5nM 2mM 3mM
Zn2+ Co2+ Ni2+ Fe3+
For [Co(H2O)6]2+
+ 3/5 o
- 2/5 o
LFSE = -5(2/5 o) + 2(3/5 o) = -4/5 o+ 2P (small)
= -7440 cm-1 (since  o = 9300 cm-1) = -21.3 kcal mol-1
For [Co(Cys)2(His)2]
+ 2/5 t
- 3/5 t
LFSE = -4(3/5 t) + 3(2/5 t) = -6/5 t + 2P (small)
= -5880 cm-1 (since  t = 4900 cm-1) = -16.8 kcal mol-1
Thus Co2+ loses 4.8 kcal mol-1 in going from aqueous solution
2+ does not.
to the zinc finger environment; Zn
Metal Folding of Biopolymers
PRINCIPLES:
•Metal-mediated protein structure changes affect transcription
•Metal-mediated protein structure changes affect translation
•Metal-induced protein structure changes also activate enzymes
•Metal-induced DNA structure changes effect anticancer activity
ILLUSTRATIONS:
•Ca2+, a second messenger and sentinel at the synapse
•Cisplatin, an anticancer drug
Roles of Calcium in Protein Folding and Activation
Calcium at the synapse: binds to the C2 domains of
synaptotagmin and helps to control neurological signal
transduction. Synaptotagmin is the sensor for Ca2+ influx.
Calcium also binds to calmodulin. As Ca2+ levels in the cell
increase, calmodulin senses these changes and binds to its
target proteins with Kd values of 10 - 100 nM or less. The
following are amino acid sequences from activated
proteins:
Synaptotagmin
is a calcium sensor
Synaptotagmin III with Mg2+
C2A-C2B crystal structure
Brunger and coworkers 1999
How does Ca2+ activate
synaptotagmin?
10-100 M Ca2+
Phospholipid
Binding
mM Ca2+
Binding to
protein
partner
(syntaxin)
Dimerization
via C2
domains?
Change in
Conformation?
Electrostatic
Switch?
FRET
340
280
C2A
C2B
Ca2+
280
C2A
470
C2B
Addition of Calcium Results in an
Increase in Energy Transfer
apo
290
mM
Ca2+
390
490
590
wavelength (nm)
7
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6
 
5

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
4 
3 
2 


1 
0
0
0.01 0.02 0.03
Concentraton of Calcium (M)
Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 5883-5888.
Other metal ions that can promote fusion
also confer this conformation change,
but to a lesser extent than does Ca2+
7
6
5

 
4




3



2
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
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
    










1      



0
0
0.002 0.004 0.006
Concentration of Divalent Metal (M)
 Ca2+ ratio
 Ba2+ ratio
 Mg2+ ratio
 Sr2+ratio
Garcia, R. A.; Forde, C. A.; Godwin, H. A. Proc. Natl. Acad. Sci. U.S.A.
2000, 97, 5883-5888.
Calmodulin - Properties and Structure
Properties: 148 amino acids; binds up to four Ca2+ ions. Primary
structure conserved over many species. Binds calcium in pairs of
“EF hand” domains. Calcium coordinated by 3 monodentate Asp,
1 bidentate Glu, 1 peptide backbone carbonyl, and 1 water
molecule. Seven-coordination favors Ca2+ over Mg2+.
Ca2+ Kd, 10-6 to < 10-7 M
Mg2+ Kd, ~ 10-3 M
Structural Changes in Calmodulin upon Binding
to a Target Peptide Domain
Note how the target
peptide is
encapsulated
following
conformational
changes in the long
central helix
A Fluorescent Sensor for Calcium Binding in Cells
Can be used to
correlate with
synaptotagmin as well
as calmodulin
response to Ca2+
levels
Summary - Points to Remember
•In eukaryotes iron homeostasis is achieved by regulating
the concentrations of the transferrin receptor and
apoferritin at the translational level. IRP’s are the
conformational switch and utilize an Fe4S4 cluster to alter
protein structure.
•A mercury resistance operon in bacteria detoxifies R2Hg
by lyase and reduction to Hg(0) which volatilizes
harmlessly out of the cell. MerR is the sensor which
undergoes a conformational change upon Hg binding and
activates the genes at the level of transcription. This topic
was not covered in class, but please read Chapter 6,
Section 6.3.
Summary - Points to Remember, cont’d
•Zinc fingers form upon metal-binding to the apo peptide
domains and activate transcription of thousands of genes.
Evidence is accumulating that zinc-binding domains also
act as metalloregulators in response to zinc levels in the
cell.
•Synaptotagmin C2 domains are the calcium sensors at the
synapse responsible for Ca-dependent signal transduction
in helping to achieve synaptic plasticity.
•Calmodulin alters its structure upon calcium influx into
cells and activates many genes at the transcriptional level.
Calmodulin allows calcium to serve as a second
messenger in response to signals at the cell surface,
affording a signal transduction pathway.
What Do These People Have in Common?
Shelley
30
Schumann
39
Keats
26
Chopin
39
Heine
59
Nietsche
56
Emily Brontë
22
Gauguin
55
Ann Brontë
29
D.H. Lawrence
45
George Orwell
47
Syphilis
Bacterial infection
Sir Alexander Fleming (1881 - 1955),
a Study in Serendipity
1922 - While suffering from a cold, Fleming made a culture from his own nasal
secretions. As he examined the culture dish, filled with yellow bacteria, a tear fell
from his eye into the dish. The next day he found a clear space where the tear had
fallen: the tear caused lysis of the bacteria but was harmless to human tissue. The
antibiotic enzyme responsible was named lysozyme, but was of little practical
importance since the bacteria were relatively harmless.
1928 - While working on cultures of influenza grown in petri dishes, Fleming
noticed an unusual clear area in one dish. Further examination revealed a bit of
mold that had fallen into the dish. Recalling his lysozyme experience, he concluded that the mold produced something deadly to the staphylococcus bacteria in
the culture dish. Fleming isolated the mold and named the antibiotic substance it
produced penicillin.
O
Penicillin: inhibits cell wall synthesis in bacteria
cells leak H2O and literally explode!
HN
S
R
O
N
-lactam ring
CO2H
Paul Ehrlich: Discovery of a Treatment for Syphilis
605 organoarsenic compounds screened before # 606 was
discovered. Actually a pro-drug:
HO
As
As
H2N
OH
NH2
pro-drug, Salvarsan
the first "magic bullet"
in vivo
O
As
OH
NH2
toxic agent
Development of Anticancer Drugs
1. Random Screening:
Folk medicine
Sea life: plants, animals - natural product chemistry
Activity
structure/function relationships
2. Specificity - the Key to Rational Design
Chemoreceptor Idea (Paul Ehrlich)
Questions: Can we design compounds by
understanding how the best agents work?
Can we harness serendipity by modern
combinatorial synthetic methods?
Factors Associated with Antitumor Drug Development
A. Serendipity: > 7000:1 in most screens
B. Perseverance: ~ 13 years between laboratory and
pharmacy
C. Cost: > $750 M
D. Moral questions: gene therapy
E. The aversion to metal-based pharmaceuticals
Cisplatin: From DNA Damage
to Cancer Therapy
Idea
Creativity
Serendipity
Understanding
& Invention
Intelligence
Discovery
Platinum Antitumor Drugs
1845
1965
1969
1978
2000
Active
H3N
Cl
Pt
H3N
Cl
cisplatin
H3N
O
O
Pt
H3N
Peyrone's chloride
Inhibition of cell growth observed
Demonstration of antitumor activity
FDA approval for genitourinary tumors
Clinical use for many types of cancer
The Cisplatin Family
Two cis amine ligands
Neutral Pt(II) or Pt(IV) compounds
Two cis leaving groups
Inactive
H 3N
Cl
Pt
Cl
NH3
NH
NH2
Pt
NH2
Cl
O
carboplatin
O
O2CR
H3N
Cl
Pt
CyH2N
Cl
RCO2
oral analogue
H3N
H2
N
Cl
Pt
N
H2
Cl
Cisplatin: An Extraordinary Anticancer Drug
100
Cl
Pt
H3N
Cl
Cisplatin
cis-diamminedichloroplatinum(II)
80
% Survival
H3N
w/ cisplatin
60
40
w/o cisplatin
1994 overall
1970 stage I
1970 stage II
1970 stage III
20
0
0
1
2
3
4
5
Years after Orchidectomy
source: I. Damjanov, Scientific American,
1995
•Cisplatin is highly effective against testicular cancer. It is also used to treat
head, neck, ovarian, and several other types of human tumors.