CALCULATION OF RELATIVE BINDING FREE-ENERGIES AND CONFIGURATIONAL ENTROPIES - A STRUCTURAL AND THERMODYNAMIC ANALYSIS OF THE NATURE OF NONPOLAR BINDING OF THROMBIN INHIBITORS BASED ON HIRUDIN(55-65)

Free energy calculations were carried out on a series of exosite-bindi ng inhibitors of thrombin. These inhibitors are based on the C-termina l fragment of hirudin and have the sequence Phe-Glu-Glu-Ile(H59)-Pro-G lu-Glu-Tyr-Leu, where the superscript over lie indicates its relative position in the natural sequence of hirudin. In this study, the effect of replacing Ile(H59) with ten other non-polar amino acids was examin ed. Three preferred interaction sites for methyl/methylene groups for the various Xaa(H59) side-chains in the complex were identified from c onformational search calculations. The corresponding thermodynamic cha nges were determined using a combination of systematic search and ener gy minimization in a manner that locates the local minima in the syste m and in the process simultaneously builds up the partition function. The free energy, internal energy and entropic contributions are readil y calculated from the partition function. Very good agreement in the r esulting relative binding free energies was obtained between theory an d experiment. The calculations allowed us to dissect out the enthalpic , entropic and solvation contributions to Delta Delta G. The contribut ion from desolvation was found to be relatively weak. The binding of t hese non-polar side-chains to thrombin is found to be driven mainly by favorable protein-ligand interactions rather than by the desire for n on-polar groups to be desolvated. We also find that the configurationa l entropy contributes about 0.48 kcal/mol (0.81 kT) in average for eac h torsional angle ''frozen'' in binding. (C) 1995 Academic Press Limit ed