STRUCTURAL CHARACTERIZATION OF THE PHOSPHOTYROSINE BINDING REGION OF A HIGH-AFFINITY SH2 DOMAIN-PHOSPHOPEPTIDE COMPLEX BY MOLECULAR-DYNAMICS SIMULATION AND CHEMICAL-SHIFT CALCULATIONS

Three molecular dynamics simulations of the free, phosphate ion-bound and phosphopeptide-bound C-terminal SH2 domain of phospholipase C-gamm a l (PLCC . pY) have been performed to aid in the interpretation of ch emical shift data and the elucidation of interatomic interactions at t he phosphotyrosine (pTyr) binding region. The simulation of the phosph opeptide complex was carried out with newly developed CHARMM force-fie ld parameters for pTyr, optimized against experimental data and ab ini tio calculations. The lack of NOEs involving phosphate in the binding pocket had necessitated a chemical shift analysis of the pTyr binding region for a more detailed characterization of the hydrogen bonding in teractions involving pTyr. Although most of these interactions are not present in the NMR structure used as the simulation starting point, t he system converges early in the simulation to a structure more compat ible with the chemical shift data. This is supported by ab initio dete rmination of the H-1(eta) and H-1(epsilon) chemical shifts of the thre e arginines (Arg 18, 37, and 39) in the pTyr binding pocket based on t he PLCC . pY MD structure, which are in accord with the experimental v alues. The simulation structure of the PLCC . pY complex reveals a mor e complete picture of interatomic interactions in the pTyr binding poc ket than is possible with current chemical shift and NOE approaches al one, thereby permitting the identification of the primary pTyr-recogni tion residues. This pattern of interactions is strikingly similar to t hose of crystal structures of related SH2 domains. The simulations als o suggest several alternative interpretations of the chemical shift da ta to those suggested in the experimental investigation (Pascal, S. M. , et al. Biochemistry 1995, 34, 11353). This insight is valuable as th e observed chemical shifts could result from a number of possible pict ures of interactions. The present study demonstrates that the combinat ion of molecular dynamics simulations and ab initio chemical shift cal culations can enhance the hydrogen-bonding, amino-aromatic, and alipha tic-aromatic information content of NOE- and chemical-shift-based prot ein structures and serve as a complementary tool for the interpretatio n of chemical shift data at the atomic level.