A REFINED MODEL OF THE THYROTROPIN-RELEASING-HORMONE (TRH) RECEPTOR-BINDING POCKET - NOVEL MIXED-MODE MONTE-CARLO STOCHASTIC DYNAMICS SIMULATIONS OF THE COMPLEX BETWEEN TRH AND TRH RECEPTOR

Previous mutational and computational studies of the thyrotropin-relea sing hormone (TRH) receptor identified several residues in its binding pocket [see accompanying paper, Perlman et al. (1996) Biochemistry 35 , 7643-7650]. On the basis of the initial model constructed with stand ard energy minimization techniques, we have conducted 15 mixed mode Mo nte Carlo/stochastic dynamics (MC-SD) simulations to allow for extende d sampling of the conformational states of the ligand and the receptor in the complex. A simulated annealing protocol was adopted in which t he complex was cooled from 600 to 310 K in segments of 30 ps of the MC -SD simulations for each change of 100 K. Analysis of the simulation r esults demonstrated that the mixed mode MC-SD protocol maintained the desired temperature in the constant temperature simulation segments. T he elevated temperature and the repeating simulations allowed for adeq uate sampling of the torsional space of the complex with successful co nservation of the general structure and good helicity of the receptor. For the analysis of the interaction between TRH and the binding pocke t, TRH was divided into four groups consisting of pyroGlu, His, ProNH( 2), and the backbone. The pairwise interaction energies of the four se parate portions of TRH with the corresponding residues in the receptor provide a physicochemical basis for the understanding of ligand-recep tor complexes. The interaction of pyroGlu with Tyr106 shows a bimodal distribution that represents two populations: one with a H-bond and an other without it. Asp195 was shown to compete with pyroGlu for the H-b ond to Tyr106. Simulations in which Asp195 was interacting with Arg283 , thus removing it from the vicinity of Tyr106, resulted in a stable H -bond to pyroGlu. In all simulations His showed a van der Waals attrac tion to Tyr282 and a weak electrostatic repulsion from Arg306. The Pro NH(2) had a strong and frequent H-bonding interaction with Arg306. The backbone carbonyls show a frequent H-bonding interaction with the OH group of Tyr282 and strong, often multiple, interactions with Arg306. Three structures, which maintained these interactions simultaneously, were selected as candidates for ligand-receptor complexes. These show persistent interactions of TRH with Ile109 and Ile116 in HX3 and with Tyr310 and Ser313 in HX7, which will be tested to refine the structure of the ligand-receptor complex. The superposition of the three struct ures shows the extent of structural flexibility of the receptor and th e ligand in the complex. The backbone of TRH inside the receptor is in an ex-helical conformation, suggesting that the receptor, through its interaction with the ligand, provides the energy required for the con formational change in the ligand from an extended to the folded form.