Backbone dynamics of the C-terminal SH2 domain of the p85α subunit of phosphoinositide 3-kinase:: Effect of phosphotyrosine-peptide binding and characterization of slow conformational exchange processes

The backbone dynamics of the C-terminal SH2 domain from the regulatory subunit F85 alpha (p85 alpha C-SH2) of phosphoinositide 3-kinase has been investigated in the absence of, and in complex with, a high-affinity phosphotyrosine-containing peptide Ligand derived from the platelet-derived growth-factor receptor. N-15 X-1 and R-2 relaxation rates and steady-state [H-1]-N-15 NOE values were measured by means of H-1-N-15 correlated true-dimensional methods and were analyzed within the framework of the model-free formalism. Several residues in the BC loop and in the neighbouring secondary structural elements display fast local dynamics in the absence of phosphotyrosine peptide ligand as evidenced by below-average [H-1]-N-15 NOE values. Furthermore, residue Gln41 (BC3) displays conformational exchange phenomena as indicated by an above-average R-2 relaxation rate. Upon binding of the phosphotyrosine peptide, the NOE values increase to values observed fur regular secondary structure and the exchange contribution to the R-2 relaxation rate for Gln41 (BC3) vanishes. These observations indicate a loss of backbone flexibility upon ligand binding. Substantial exchange contributions fur His56 (beta D4) and Cys57 (beta D5), which are known to make important interactions with the ligand, are attenuated upon ligand binding. Several residues in the beta D'-FB region and the BG loop, which contribute to the ligand binding surface of the protein, exhibit exchange terms which are reduced or vanish when the ligand is bound. Together, these observations suggest that ligand binding is accompanied by a loss of conformational flexibility on the ligand binding face of the protein. However, comparison with other SH2 domains reveals an apparent lack of consensus in the changes in dynamics induced by ligand binding. Exchange rates for individual residues were quantified in peptide-complexed F85 alpha C-SH2 from the dependence of the exchange contributions on the CPMG delay in an R-2 series and show that peptide-complexed F85 alpha C-SH2 is affected by multiple conformational exchange processes with exchange rate constants from 10(2) s(-1) to 7 . 10(3) s(-1). Mapping of the exchange-rate constants on the protein surface show a clustering of residues with similar exchange-rate constants and suggests that clustered residues are affected by a common predominant exchange process. (C) 2000 Academic Press.