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−γ1 (PLCC·pY) have been performed to aid in the interpretation of chemical shift data and the elucidation of interatomic interactions at the phosphotyrosine (pTyr) binding region. The simulation of the phosphopeptide complex was carried out with newly developed CHARMM force-field parameters for pTyr, optimized against experimental data and ab initio 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 interactions involving pTyr. Although most of these interactions are not present in the NMR structure used as the simulation starting point, the system converges early in the simulation to a structure more compatible with the chemical shift data. This is supported by ab initio determination of the 1Hη and 1Hε chemical shifts of the three arginines (Arg 18, 37, and 39) in the pTyr binding pocket based on the PLCC· pY MD structure, which are in accord with the experimental values. The simulation structure of the PLCC·pY complex reveals a more complete picture of interatomic interactions in the pTyr binding pocket than is possible with current chemical shift and NOE approaches alone, thereby permitting the identification of the primary pTyr-recognition residues. This pattern of interactions is strikingly similar to those of crystal structures of related SH2 domains. The simulations also suggest several alternative interpretations of the chemical shift data to those suggested in the experimental investigation (Pascal, S. M., et al. Biochemistry 1995, 34, 11353). This insight is valuable as the observed chemical shifts could result from a number of possible pictures of interactions. The present study demonstrates that the combination of molecular dynamics simulations and ab initio chemical shift calculations can enhance the hydrogen-bonding, amino−aromatic, and aliphatic−aromatic information content of NOE- and chemical-shift-based protein structures and serve as a complementary tool for the interpretation of chemical shift data at the atomic level.