Abstract
Recent advances in NMR methodology have made it a powerful approach for the study of biomolecular structure and dynamics (Bax and Grzesiek, 1993; Bax, 1994; Farrow et al., 1994a). In addition to NMR being a structural tool, as a solution spectroscopy it is exquisitely sensitive to dynamic processes - not only fast, low amplitude motions which can often be described by analysis of X-ray crystallographic B factors (Ringe and Petsko, 1985), but also slower, larger amplitude motions. These may include conformational exchange on millisecond time-scales or longer between states as dissimilar as folded and unfolded states of proteins and reflecting motions of tens of angstroms. We have exploited this distinguishing capability of NMR spectroscopy to describe the dynamic processes observed during structural studies of two isolated domains of signal transduction proteins, a Src Homology 2 (SH2) domain of phospholipase Cγ in complex with a phosphopeptide from the platelet-derived growth factor receptor (PDGFR) and an isolated Src Homology 3 (SH3) domain from the drosophila protein Drk1.
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Discussion
Weixing Zhang - If you mutate the position +4 to +6 of your phosphopeptide, what is the difference in SH2 binding compared to non-mutated phosphopeptide?
Foman-Kay - It is important to realize that you cannot make generalizations from one SH2 complex to another and I think that this is one of the significant points that has been emerging. It is difficult to classify SH2 domains and to say that for this class only certain residues are important for binding while for a second class other residues are important. It’s necessary to do binding studies and specific truncation studies or alanine scans in order to get at the data for each individual system. Our collaborator, Steve Shoelson, has done some very interesting work where he has shown that you can actually remove all of the residues other than the -1, pTyr and +1 positions (Le. removing +2 and beyond) and still
maintain a significant binding affinity in the case of C-terminal SH2 of PLCγ, whereas you absolutely need positions +2, +3 and +4 for binding in the structurally similar Syp phosphatase N-terminal SH2 domain. However, our collaborator Tony Pawson has done studies showing that if you change the +4 position in the P’Y 1021 peptide, which is a leucine, to a serine, then PLC’YSH2 will bind but in addition the SH2 domain of the p85 subunit of P13K will bind. Then, if you make an additional substitution of the +3 position to a methionine, you can convert the peptide so that p85 is the only SH2 domain that will bind and the PLC’YSH2 does not bind. What I am saying is that there are no generalizations and mutagenesis studies and specific binding studies have to be done in each case; it’s complicated. We’re trying to understand this complexity.
Zhang - Thank you.
Marc Adler - The folding data suggested that there are two models which could potentially explain the data. Could the non-cooperativity be explained by there being a third state? You analyzed it according to a two state model. Could there be a third state which is in rapid equilibrium with an “unfolded” one and could that explain the various rates? Would that be consistent with the data?
Forman-Kay - I think it would. It would be impossible for us to confirm, however, if that third state is in rapid exchange with the folded state or the unfolded state. It’s impossible to actually get an experimental handle on it, but it’s a model which we are pursuing to try to understand the current data.
Adler - Another one, I’ll try to keep it quick. You said the N-N connectivities suggest the α. region of the φ - ψ space. Could you have a local formation of an oil drop which included a lot of tight turns and therefore gave rise to the N-N connectivities? This could be exchanging rapidly.
Forman-Kay - When I say that the region of conformational space is preferentially sampled, I am not talking about a cooperative formation of helices at all. I am talking about local helical turns. You can call them B turns or helical turns but that region of conformational space which includes turn conformations on a local level is what’s being sampled. I don’t think there’s any cooperativity between the individual residues. I don’t think there is a formation of an actual helix.
Adler - Thank you.
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Forman-Kay, J.D. et al. (1996). Structural, Dynamic, and Folding Studies of SH2 and SH3 Domains. In: Rao, B.D.N., Kemple, M.D. (eds) NMR as a Structural Tool for Macromolecules. Springer, Boston, MA. https://doi.org/10.1007/978-1-4613-0387-9_3
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