Abstract
The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA) to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the “two-pronged plug two-hole socket” model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an α-helix. In contrast, a β-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.
Highlights
SH2 (Src homology 2) domains are found as modules in many proteins involved in cell signal transduction pathways
The deviations from the extended conformation observed in the SH2-bound peptides pYEEA and pYEEG are similar to those reported in a more sophisticated, parallel tempering dynamics study of Ala and Gly mutants of tional trends of phosphopeptides complexed with SH2 domains
The results of the fluorescence polarization assays suggest that the two glutamic acids at positions phosphorylated tyrosine residues (pTyr)+1 and pTyr+2 provide a significant contribution to the binding energy through their electrostatic interactions with positively charged residues on the surface of the Src SH2 domain, and that all three residues
Summary
SH2 (Src homology 2) domains are found as modules in many proteins involved in cell signal transduction pathways They bind to short stretches of amino acids that contain phosphorylated tyrosine residues (pTyr) and thereby mediate the interactions between proteins during cell signaling. Development of efficient small-molecule inhibitors has proven difficult, suggesting that a better understanding of the structural basis underlying phosphopeptide-SH2 domain interactions is required To this end we have undertaken a computational and experimental study to systematically evaluate the role residues C-terminal to the pTyr anchor may play in the binding affinity and conformation of phosphopeptides when bound to the Src SH2 domain
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