Abstract
Grb2, a modular protein comprised of a central SH2 domain flanked between a N-terminal SH3 (nSH3) domain and a C-terminal SH3 (cSH3) domain, is a component of cell signaling networks involved in the transmission of extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. The Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites - designated S1, S2, S3, and S4 - containing PXpsiPXR motifs within Sos1. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry coupled with molecular modeling and semi-empirical analysis, I provide new insights into the Grb2-Sos1 interaction in thermodynamic and structural terms. My data show that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven. The heat capacity change observed was much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon dimerization, implying that monomers undergo conformational rearrangement upon dimerization. 3D structural models suggest strongly that such conformational rearrangement may arise from domain swapping. I further show that the nSH3 domain of Grb2 binds to the S1 site containing the proline-rich consensus motif PXpsiPXR with an affinity that is nearly three-fold greater than that observed for the binding of the cSH3 domain. It is also demonstrated that such differential binding of the nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue, in the RT loop, to engage in the formation of a salt bridge with the arginine residue in the consensus motif PXpsiPXR. The data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Additionally, my data reveal that while the nSH3 domain of Grb2 binds with affinities in the physiological range to all four sites S1-S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3 and S4 sites, the PXpsiPXR motif at S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site but their role is not critical for the recognition of S2, S3 and S4 sites. Molecular modeling is employed to rationalize my new findings in structural terms. Taken together, this thesis provides novel insights into the physicochemical basis of a key protein-protein interaction pertinent to cellular signaling and cancer. My studies bear the potential for the development of novel therapies with less toxicity but more effectiveness for the treatment of disease.