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Transcript
Biophysics 204 Discussion Papers
Robert Fletterick June 28, 2017
PROTEIN-PROTEIN INTERACTIONS
The questions that I present here are provided to encourage discussion including
background and detailed resolution about the point if appropriate.
Read Jan 7, discuss in class Jan 9
Paper 1. Wilson JJ, Kovall RA. Crystal structure of the CSL-Notch-Mastermind ternary
complex bound to DNA. Cell. 2006 Mar 10;124(5):985-96.
Protein interaction energies are not predictable; still a framework to discuss the principles is
important. When complexes contain more than two molecules that come together for function the
interactions rationalize the biological control systems. One of the most studied and complicated
signaling systems uses Notch receptors to pass developmental signals between neighboring cells.
A protein-DNA complex assembles on a promoter that leads to transcription of target genes. The
manuscript is one of two in this issue of Cell that show the crystal structure of a part of a Notch
transcriptional complex containing the ankrin domain of Notch (ANK), the transcription factor CSL,
a polypeptide from the coactivator Mastermind assembled on target DNA. The elaborate interaction
surfaces restrict the recruitment of proteins to promoters ensuring transcriptional control of Notch
target genes.
What the paper is aboutThis paper presents a structure of a part on a set of elaborate molecular machines that ready
chromatin for copying to RNA. This is a multi step process, with enzymology, and with many
interaction molecules that come together in three dimensions. In eukaryotic transcription, there are
two principle active processes; activation of transcription, and the second is repression. Both
require enormous changes since eukaryotic DNA is found in packages with covalently attached
chemical markers, methyl, acetyl, phosphate, and access to it can be highly regulated.
Important Bonus!
Please read: Direct inhibition of the NOTCH transcription factor complex. Moellering RE,
Cornejo M, Davis TN, Del Bianco C, Aster JC, Blacklow SC, Kung AL, Gilliland DG, Verdine GL,
Bradner JE. Nature. 2009 Nov 12;462(7270):182-8.
This shows in red an alpha-helical peptide targeting the mastermind protein that stabilizes the
NOTCH complex.
Discussion points
There is an elaborate introduction to tersely place the structure in biological context.
1. What is the question that this paper would attempt to answer?
2. What are notch, CSL and mastermind and how do they relate in function?
3. Remind us of the major steps in notch signaling.
4. How was it possible to identify the component domains that would form a stable
complex?
5. What are the functions of the three CSL domains?
6. What are the two domains of notch and what are their functions?
7. What is the background information on roles of ankrin repeats?
8. Describe the DNA interactions from the DNA perspective to show how specificity is read
and binding is achieved.
9. Mastermind proteins are not conserved across species. How does mastermind contribute
to this complex stability and how do we account for lack of conservation in the conserved
Notch signaling pathway?
10. What is the conformational change in CSL comparing this complex with CSL alone on
DNA.
11. What is used to align the models?
12. Might crystal-packing forces on CSL, or its partners, affect these conclusions?
13. Summarize and criticize the proposed mechanism of conversion of repressor to activator
suggested by the authors.
14. BONUS discussion point- Why is it difficult to drug transcription factors? Will the inhibitor
described in this paper become a drug?
Read Jan 9, Discussion on Jan 11, two papers:
The objective is to learn about experimental methods for discovering interactions between proteins
and how to measure associations. The topics are expanded in the discussion papers here to
include protein interactions in assembly of integral membrane proteins. To simplify this field, the
papers are focused on inter protein interactions between two helices found within the membrane
soluble domains of the proteins.
Call ME, Pyrdol J, Wiedmann M, Wucherpfennig KW. The organizing principle in the
formation of the T cell receptor-CD3 complex. Cell. 2002 Dec 27;111(7):967-79
Call ME, Schnell JR, Xu C, Lutz RA, Chou JJ, Wucherpfennig KW. The structure of the 
transmembrane dimer reveals features essential for its assembly with the T cell receptor.
Cell. 2006 Oct 20;127(2):355-68.
What the 2002 paper is aboutOn the surface of immune cells, the T cell receptor recognizes proteins projecting from antigen
presenting cells. The receptor core is protein chains forming a heterodimer responsible for ligand
recognition, to which are added helper dimers of CD3’s called CD3γε, CD3δε. It also contains two
zeta chains, ζζ. If the binding recognition is appropriate, the receptor becomes active and changes
the cell’s genetic program. The receptor and some of its partner proteins are membrane integrated
through a short a helix passing through the lipid bilayer. Modeling shows nine conserved, probably
charged residues in the transmembrane helices. Signaling requires associations of three signaling
dimers with the receptor. The authors developed methods for isolating protein complexes to
demonstrate that one basic and two acidic transmembrane residues are required for the assembly.
A three-helix arrangement drives the assembly.
Discussion points:
Immunoprecipitation confuses biochemistry of large multisubunit assemblies by giving both the full
complex and unassembled chains and assembly intermediates.
1. How did the authors combine tags and tag binding proteins to insure that only full molecules
were found?
2. What is the molecular stoichiometry of this complex?
3. How many transmembrane helices come together on activation?
4. Describe the three-Helix Motif in the Assembly of CD3δε with TCRα.
5. What hypothesis and experiments proved this assembly (data and experiments in Figure 2)?
6. How was the specificity for interactions of the three helices of TCRa and its CD3 dimer
shown?
7. What experiments in Figure 4 show similar specificity for linking the ζ subunit on TCR to its
CD3 dimer?
8. What is the proof that the helix triplets form the basis of specificity of assembly?
9. Referring to Fig 6 what is the data showing that the ζ chains and the Arg on the TCR found
in the transmembrane helix are essential for adding ζ chains to the complex?
10. What is the assembly mechanism?
11. How do the authors rationalize the three-helix assembly model with required fidelity for the
signaling event?
12. Referring to the 2006 paper cited above describe the interactions and specificity of the ζζTM
Dimer Interface.
13. The interface between the helices of the dimer is dominated by polar contacts, but are the
interactions of the paired Asp’s as determined here relevant in the complex?
Read Jan 14, Discuss in class January 16. Paper 3.
Fab’s are a paradigm for protein-protein interactions. Many cells communicate by forming protein
interactions through domains that belong in the immunoglobulin family. This paper presents work
intended to discover the principles employed by antibodies in tuning affinity to binding partner
antigens.
Li Y, Li H, Yang F, Smith-Gill SJ, Mariuzza RA. X-ray snapshots of the maturation of an
antibody response to a protein antigen.
Nat Struct Biol. 2003 Jun; 10(6): 482-8.
What the paper is about• Details from X-ray crystallography of a lysozyme-binding antibody
• How a binding site for a protein evolves in sequence to become stronger
• The features that nature uses to improve protein-protein dissociation constants
• Characterization of Fab antigen interfaces
Discussion points
1. What is the goal of this paper?
2. What is the background of the issues addressed?
3. How do antibodies get made in such a short time interval to evolve tight binding to
antigen?
4. What constraints are on the amino acids that Fabs may substitute to affect affinity for
antigen?
5. What constraints are on the structure of the Fab as it is being remodeled?
6. What is meant algorithmically by the shape complementarity and how is it calculated?
7. Compare the surfaces of the interfaces between antibodies.
8. What features of the lysozyme does the antibody favor as it evolves to become stronger?
9. What structural changes are observed in comparing the structures of the four
complexes?
10. How important to tight binding are the conformational changes?
11. Compare the polar and nonpolar and hydrogen bonding potentials at the four interfaces.
12. Summarize what was learned and how it is relevant to other protein interfaces.
Read Jan 16, Discussion January 18 Paper 4.
Structure of the intact PPAR-γ–RXR-α nuclear receptor complex on DNA
Vikas Chandra, & Fraydoon Rastinejad, Nature, 350-356 Published online 29 October 2008;
What this paper is about-
The paper is significant for understanding structure-function of Nuclear Receptors (NR) which
recruitment cofactors. Nuclear receptors have typically 4 domains. The central zinc finger DNAbinding domain (DBD) relates to the C-terminal hormone-binding domain (LBD) that engenders
part of hormone-dependent activation function (AF2). A hinge region links the DBD and LBD. The
N-terminal domain varies from 20 to 600 amino acids and forms AF1, a segment that may also
activate transcription. The hormone helps fold the structure of the NR, in part positioning H12
which form part of the binding site for cofactors.
The peroxisome proliferator-activated receptor (PPAR), one of the classes of NR’s that work with
the master NR called RXR, regulates fat and carbohydrate metabolism. The paper shows the
crystal structure of PPARγ-RXRα heterodimers on DNA. Two new features are found. An extra
segment of DNA upstream of the response element, 5′ extension of DR1 helps position the
complex. The hinge of PPARγ binds the extension working with the DBD. Domains not
unexpectedly form an interacting functional configuration.
1. What small and large molecules are found in the complex?
2. Given the crystals, how was the structure determined?
3. What was known about the conformations and configurations of NR hinges before this
paper?
4. Explain the concept of DNA half site the so-called DR1 element.
5. What is the structure of the DNA?
6. How do the structures of the individual domains relate to the structures of the domains in
the complex?
7. What is the RXR-α CTE?
8. How might the master NR use this CTE to bind NRs?
9. What is the symmetry relating the RXR LBD with the PPAR LBD?
10. Is there a predicted order of the DBDs and receptors on the DR1 element?
11. Are the DBD’s in contact?
12. How do the major and minor grooves of DNA engage the proteins?
13. What are the contacts and their significance of the PPAR LBD?
14. When DBD to DBD association constants are measured with DNA, the binding is weaker
that found in the full-length protein. Why?
15. Structures are shown for two hormone like drugs. Are the structures significantly
different?
16. Might other drugs change the assembly? How?
17. What effect does DNA have on protecting H-D exchange of PPAR?
18. What do the H-D exchange data suggest for role of the N terminal domains relating to
this structure?
19. Does the domain configuration in the complex suggest interactions for coregulators,
aside from simple uncooperative binding?
20. Might drug binding to a NR LBD affect DNA binding of the receptors?