Backbone dynamics of the C-terminal SH2 domain of the p85 alpha subunit ofphosphoinositide 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 subu nit F85 alpha (p85 alpha C-SH2) of phosphoinositide 3-kinase has been inves tigated in the absence of, and in complex with, a high-affinity phosphotyro sine-containing peptide Ligand derived from the platelet-derived growth-fac tor 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 structur al elements display fast local dynamics in the absence of phosphotyrosine p eptide ligand as evidenced by below-average [H-1]-N-15 NOE values. Furtherm ore, residue Gln41 (BC3) displays conformational exchange phenomena as indi cated by an above-average R-2 relaxation rate. Upon binding of the phosphot yrosine peptide, the NOE values increase to values observed fur regular sec ondary structure and the exchange contribution to the R-2 relaxation rate f or Gln41 (BC3) vanishes. These observations indicate a loss of backbone fle xibility upon ligand binding. Substantial exchange contributions fur His56 (beta D4) and Cys57 (beta D5), which are known to make important interactio ns 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 bind ing surface of the protein, exhibit exchange terms which are reduced or van ish when the ligand is bound. Together, these observations suggest that lig and binding is accompanied by a loss of conformational flexibility on the l igand binding face of the protein. However, comparison with other SH2 domai ns 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 co ntributions on the CPMG delay in an R-2 series and show that peptide-comple xed F85 alpha C-SH2 is affected by multiple conformational exchange process es with exchange rate constants from 10(2) s(-1) to 7 . 10(3) s(-1). Mappin g of the exchange-rate constants on the protein surface show a clustering o f residues with similar exchange-rate constants and suggests that clustered residues are affected by a common predominant exchange process. (C) 2000 A cademic Press.