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Module Code: BIO00025H
Examination Candidate Number: __________
Desk Number: __________
BA, BSc, and MSc Degree Examinations 2017-8
Department : BIOLOGY
Title of Exam: Protein-protein recognition
Time Allowed:
2 hours
Marking Scheme: Total marks available for this paper: 100
Sec�on A: Short Answer / Problem / Experimental Design ques�ons (50 marks) Sec�on B: Essay ques�on (marked out of 100, weighted 50 marks) The marks available for each ques�on are indicated on the paper
Instructions:
Sec�on A: Answer all ques�ons in the spaces provided on the examina�on paper Sec�on B: Answer either ques�on A or ques�on B. Write your answer on the separate paper provided and a�ach it to the back of the ques�on paper using the treasury tag provided.
Materials supplied: CALCULATOR
For marker use only: For office use only:
1 2 3 4 Module total as %
DO NOT WRITE ON THIS BOOKLET BEFORE THE EXAM BEGINS
DO NOT TURN OVER THIS PAGE UNTIL INSTRUCTED TO DO SO BY AN INVIGILATOR
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Module Code: BIO00025H
SECTION A: Short Answer / Problem / Experimental Design questions
Answer all questions in the spaces provided
Mark total for this section: 50
1. a) Explain the terms core and rim when referring to residues in protein interfaces and comment on why these definitions are helpful in characterising protein-protein interaction sites. (3 marks)
A core residue contributes to the interface and has one or more atoms that are completely buried by the formation of the complex [1]. A rim residue contributes to the interface but has no atoms that are completely buried upon formation of the complex [1]. The core definition reveals interface residue propensities which are not apparent if the surface as a whole is considered [1]. Most students could define core and rim though fewer could articulate why these terms are helpful.
b) Explain the term hydrophobic effect and explain its contribution to protein-protein interactions. (4 marks)
The hydrophobic effects derives from the disruption to the dynamic hydrogen bonding structure of water that occurs at an apolar interface. The coming together of apolar surfaces to form an interface reduces the area of exposure of apolar surface to water. The ‘liberated’ (more disordered) water forms more favourable ‘disordered’ structure making a positive contribution to deltaS and therefore a negative change in delta G. This change in the free energy of the water gives the hydrophobic effect. Most students scored reasonable marks on this question. In this type of question, the answer should refer to residues as apolar rather than hydrophobic to avoid circular argument. The contribution of water was not always emphasised sufficiently. 2. a) Two monomeric proteins A and B form a complex with 1 : 1
stoichiometry and a K d of 3.0 x 10 –7 M. After mixing 16 μM A with B, it is found that the equilibrium concentration of the free A is 6 μM. Showing your reasoning, calculate the overall concentration of B in the mixture.
(5 marks) If the free A concentration is 6 μ M, the concentration in the complex [A-B] = 16 μ M -
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6 μ M = 10 μ M. [1]
K d = [A].[B]/[A-B] [1] => 3 x 10 –7 M = (6 μ M.[B])/10 μ M [1]
=> [B] = (3 x 10 –7 M)(10 x 10 –6 M) /(6 x 10 –6 ) = 0.5 x 10 –6 M [1]
=> [B]tot = [B] + [A-B] = 0.5 x 10 –6 M + 10 μ M = 10.5 μ M [1]
A small number of students scored full marks on this ques�on but a disappoin�ngly large number of students were unable to formulate an answer and did not get much further than wri�ng the equa�on for Kd.
b) The Figure below shows structures of SpoIIQ-SpoIIIAH and gp120-CD4-F ab complexes. Comment on the structural characteristics of the protein-protein interfaces and their functional significance.
(4 marks)
( Left) The interfaces here involve multiple loops involving immunoglobulin domains.
The gp120-CD4 interaction contributes to immune cell recognition/infection by HIV
while the Fab-gp120 interaction is involved in protection against the virus [2].
(Right) The interface here involves an intermolecular beta-sheet. These proteins
form an intercellular complex involved in membrane migration and later an
intercellular channel. [2].
This question was done badly and only a minority of students distinguished the complexes correctly.
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Module Code: BIO00025H
3. Research on protein A associated with a rare genetic disorder established the following protein-protein linkages:
Pair of proteins
Linkages
A, B gene fusion immunoprecipitation
B,C gene co-occurrence negatome database
C,D isothermal titration calorimetry surface plasmon resonance cryo-EM structure
E,A gene neighbourhood gene fusion
F,A analytical ultracentrifugation surface plasmon resonance yeast two-hybrid
G,A analytical ultracentrifugation gene neighbourhood yeast two-hybrid
a) Showing only possible interacting partners, draw a schematic
diagram for the network formed by protein A , indicating interactions as shown below:
Physical, established in one-to-one experiments (solid line) Physical, established by proteome-wide analysis (dash dot line) Functional interactions (dot line) (6 marks)
1 mark for the correct set of interacting proteins and 0.5 mark for each correctly
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assigned interaction
Most student performed well, but only few scored full marks.
b) Rank interacting partners of protein A, starting from the least likely interacting partner and finishing with the most likely. (3 marks)
1 mark for identifying the least likely, 1 mark for identifying the most likely partner and 1 mark for the correct order E,B,G,F Performance on this question was mixed.
c) Name two linkages of protein A that were established by proteome-wide methods. (2 marks)
Immunoprecipitation, yeast two-hybrid There was a small proportion of students who mixed up “linkage” with an interaction partner and several who did not identify correct linkages.
d) Name two linkages of protein A that were established in vitro . (2 marks)
AUC, SPR There was a small proportion of students who mixed up “linkage” with an interaction partner and several who did not identify correct linkages.
e) Further studies revealed that protein A is a major protein interaction hub, with its N-terminal and C-terminal domains involved in “one-to-many” and “many-to-one” types of interaction, respectively. Briefly outline the structural basis for each type of interaction giving an example from lecture material. (4 marks)
For each type of interactions: 1 mark for explaining the structural basis and 1
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Module Code: BIO00025H
mark for a suitable example. One-to-many: mediated by protein segments that can adopt different conformations upon interaction with binding partners. A good example would be the p53 protein: a short segment at its C-terminus can attain a helical, beta-strand or a distinct irregular structure upon interaction with S100b, Sirtuin, Cyclin A2 or CBP. Many-to-one: the same binding site on protein’s surface can accommodate segments from multiple partners. For example, the 14-3-3 protein can bind a selection of phosphopeptides present in intrinsically disordered regions of partner proteins. Mixed performance: although a number of students scored full marks, many students either didn’t explain the structural basis and/or couldn’t identify correct examples.
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4. The table below presents protein binding affinities measured in a series of isothermal titration calorimetry experiments in which different fragments of microtubule plus-end tracking proteins were mixed.
a, no binding detected b, the data did not allow a rigorous measurement of affinity but did suggest an affinity in the millimolar range Data taken from Weisbrich et al., (2007) Nat Struct Mol Biol , 14 , 959-967
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The protein constructs used in these experiments and how they relate to full-length EB1, p150Glued and CLIP170 are shown schematically below:
a) Compare and contrast the information obtained from surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) experiments.
(4 marks)
Both techniques can give affinities of binding (1 mark); SPR can also give information about interaction kinetics (1 mark); ITC can give thermodynamic information (dG, dS, dH) (1 mark) and stoichiometry (1 mark).
Unfortunately, a number of people missed the emphasis in the question on the information obtained from the techniques and therefore many answers focused on irrelevant practical details, e.g. protein concentration, proteins attached to chips, etc.
b) How does mutation of the acidic tail of EB1 impact binding to p150Glued. Use the data in the table and your knowledge of CAP-Gly domains to justify your answer. (4 marks) Question refers to three mutants of EB1c and their interaction with p150n. Mutation of EB1c Tyr-268 to Phe does not alter binding affinity compared to WT, both have Kd ~ 2.4 uM (1 mark). Mutation of Tyr-268 to either Ile or Gly reduces binding 10-15 fold (1 mark). Therefore an aromatic residue is required the C-terminus of EB1 (1 mark). The
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Module Code: BIO00025H
aromatic side chain at position 268 binds to an aromatic pocket in the CAP-Gly domain of p150Glued (1 mark)
On the whole, answered fairly well but many people failed to state that the aromatic ring of an acidic tail is important because it is recognised by an aromatic pocket in the CAP-Gly domain. This answer could have been obtained from the data in the table even without knowledge of the CAP-Gly domain structure. The GKNDG motif principally interacts with the negatively charged carboxylate group at the end of the EEY polypeptide, not the aromatic ring of the C-terminal residue.
c) Suggest why ClipZn1 does not interact with p150n while ClipZn2 does (1 mark)
ClipZn1 lacks a C-terminal acidic tail motif Several answers were unnecessarily complicated.
d) What do the effects of mutation of residues 68, 69, 90 and 93 of p150n tell you about the interaction with ClipZn2. (3 marks) Question refers to 8 mutants of p150n and their interaction with ClipZn2. Mutations that remove positively charged residues (e.g. K68A, R90P or R90S) substantially reduce binding affinity (1 mark) while mutations that introduce a positive charge (e.g. Q93K or Q93R) enhance binding affinity (1 mark). These data suggest that these residues interact with a region or motif of ClipZn2 that is negatively charged (1 mark)
It is important to know that an increase in the affinity of an interaction means the value of the Kd for that interaction decreases, and vice-versa. Affinity is a descriptive and subjective term (e.g. weak, intermediate, strong, low, high, etc), while Kd (dissociation constant) is numerical and quantitative (e.g. 10 nM, 1 mM, 4.5 × 10 -6 M, etc). An interaction with a Kd of 10 nM (1 × 10 -8 M) has a higher affinity than one with a Kd of 0.7 mM (7 × 10 -4 M). Lots of mistakes were due to misunderstanding of that fundamental concept, here and throughout Q4. It is also important to note how the residue substitutions change the chemistry, relate that to the observations in the table (e.g. mutations that remove a positive charge decrease affinity while those that add a positive charge increase affinity), state that in the answer, and speculate how that observation may be linked to interface composition and recognition.
e) CLIP170 has been shown to form a closed, inhibited state in which
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ClipCG1 and ClipCG2 domains interact with the Zn12 region. Predict what might happen if p150Glued is added to the closed form of CLIP170. Use the data in the table to justify your answer. (2 marks) p150n binds Zn12 3-6 times more strongly than the CG domains of CLIP170 bind Zn1 or Zn2 (1 mark). p150n would likely displace the CG domains relieving the closed state of CLIP170 (1 mark). Generally answered well. f) The EB1c construct forms a homodimer in solution. Explain the binding affinities measured in ITC experiments involving EB1c and ClipCG1, ClipCG2 and ClipCG12. (3 marks) Each acidic tail motif in the EB1c homodimer can interact with a Clip CG domain (1 mark). The tighter binding of the double domain ClipCG12 construct is due to weak avidity (1 mark) because the two CG domains are on the same molecule (1 mark) More difficult than anticipated. No-one mentioned avidity.
LO - explain how modular protein structures and common domains and motifs contribute to plus-tip interaction networks LO - appreciate the type of quantitative data that can come from biophysical studies of physical protein/protein interactions and how and when to use these approaches
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SECTION B: Essay question
Answer one question on the separate paper provided
Remember to write your candidate number at the top of the page and indicate whether you have answered question A or B
Mark total for this section: 50 EITHER A) Using appropriate examples, explain how solution NMR spectroscopy
has illuminated the role of intrinsically disordered binding motifs in protein-protein recognition.
There are several parts of the lecture material that this question could address. It is likely that most answers will include reference to the interaction between the KIX domain of CBP and the pKID motif of CREB. Other unstructured motifs that interact with KIX were also discussed in the lecture. Another lecture dealt with the C-terminal unstructured EEY/F motifs of proteins that interact with CAP-Gly domains. Rather than just describing interacting systems that feature unstructured proteins or regions of proteins, the answer should explain what was found by these studies. This could include information about the measurement of binding affinity (measurement of Kd was mentioned in the lecture), information about 3D structure (i.e. resulting from structural studies) or the location of binding sites, information about the formation of encounter complexes, etc. In all instances, examples will be needed to show how NMR was used and to what system. Disappointing. Only 6 responses and none were outstanding. Average of ~58. The highest mark (69) was a nearly error-free recital of material from Lecture 10 but presented no evidence of any of the excellence indicators and no evidence of any extra reading outside of the lecture slides. Other answers were weaker and more error strewn and contained irrelevant (e.g. GTPases) and/or poorly described material (e.g. p53 but without detailed description of relevant NMR-based studies). The lack of evidence of excellence markers was disheartening given that these were highlighted throughout the lecture.
LO - Be able to explain,using appropriate examples,how NMR spectroscopy
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Module Code: BIO00025H
can be used to investigate protein/protein recognition
LO - Understand the concept of encounter complexes (e.g.proteins that associate with KIX)
LO - Explain how the modular structure of proteins like p300/CBP contribute to their function
LO - Understand that unstructured regions of proteins can play key functional roles in protein/protein recognition
OR B) Discuss how early and late compartment-specific gene expression is
established in the forespore during sporulation in Bacillus . Refer to the roles of relevant protein-protein complexes, commenting on the techniques used to identify and characterize these complexes, and the supporting evidence for their existence.
Answer: This is an essay with a substantial amount of lecture course material to draw on. 1. The interactions of SpoIIE and the three proteins of the spoIIA operon that lead to activation of sigma F were covered at length. 2. The interactions of SpoIIQ and SpoIIIAH that lead to activation of sigma G were also discussed in detail. 3. The students can refer gel-techniques, fluorescence spectroscopy, protein crystallography as well as in vivo tagging experiments – SPR and affinity techniques were also mentioned briefly and the students could describe these in more detail drawing on other sources. A good essay is likely to set the scene in relation to asymmetric septation and alternate sigma factors as the agents of cell differentiation. They can explain how sigma factors interact with core polymerase and are responsible for promoter recognition. In the case of sigma F, introduce SpoIIAB as anti-sigma factor and SpoIIAA as its antagonist. The interaction of these proteins with sigma F and ADP/ATP can be illustrated by native gel electrophoresis and/or fluorescence spectroscopy. X-ray crystallographic description of sigma F :AB and AA:AB complexes and effect of phosphorylation of AA. SpoIIE as the AA~P phosphatase that is localised to the sporulation septum – through outside reading they may pick up on interactions of FtsZ and the divisome. Explain notion of induced release of sigma F from sigma F :AB by AA and how this was established by site-directed mutagenesis in which heterodimers were made.
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Again wider reading could lead to answers describing intricacies of heterodimer construction. The essay should include mention of the finding that the higher specific activity of SpoIIE in the forespore could be a consequence of volume asymmetry, thus accounting for all-or-nothing sigma F activation and could include recent observations on how SpoIIE is activated. The sigma G aspects of the essay should cover the process of engulfment of the forespore by the mother cell and the role of SpoIIQ and SpoIIIAH in this process. These forespore and mother cell proteins respectively form an intercellular zipper which facilitates membrane migration and later they form an channel through which a factor passes that is needed for sigma G activation. This story is less clear and evidence should be weighed. Expect to see mention of BirA and biotin-acceptor peptide labelling experiments as well as SPR, ITC, CD and crystallographic experiments. This proved to be a very popular question with 27 of the 33 candidates choosing this essay. The mean mark for the essay was 64 %. Most students showed good knowledge and understanding of the subject especially the regulation of sigma F. A weakness of some essays was extended description of the Q-AH channel without pointing out the context of this channel in the establishment of late compartment-specific gene expression in the forespore. The best essays showed a capacity to weave in the techniques used to identify and characterize these complexes, and the supporting evidence for their existence without disrupting the flow of the narrative.
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