ENZYME FLEXIBILITY, SOLVENT AND WEAK-INTERACTIONS CHARACTERIZE THROMBIN-LIGAND INTERACTIONS - IMPLICATIONS FOR DRUG DESIGN

Background: The explosive growth in the rate of X-ray determination of protein structures is fuelled largely by the expectation that structu ral information will be useful for pharmacological and biotechnologica l applications. For example, there have been intensive efforts to deve lop orally administrable antithrombotic drugs using information about the crystal structures of blood coagulation factors, including thrombi n. Most of the low molecular weight thrombin inhibitors studied so far are based on arginine and benzamidine. We sought to expand the databa se of information on thrombin-inhibitor binding by studying new classe s of inhibitors. Results: We report the structures of three new inhibi tors complexed with thrombin, two based on 4-aminopyridine and one bas ed on naphthamidine. We observe several geometry changes in the protei n main chain and side chains which accompany inhibitor binding. The tw o inhibitors based on 4-aminopyridine bind in notably different ways: one forms a water-mediated hydrogen bond to the active site Ser195, th e other induces a rotation of the Ser214-Trp215 peptide plane that is unprecedented in thrombin structures. These binding modes also differ in their 'weak' interactions, including CH-O hydrogen bonds and intera ctions between water molecules and aromatic pi-clouds. Induced-fit str uctural changes were also seen in the structure of the naphthamidine i nhibitor complex. Conclusions: Protein flexibility and variable water structures are essential elements in protein-ligand interactions. Liga nd design strategies that fail to take this into account may overlook or underestimate the potential of lead structures. Further, the signif icance of 'weak' interactions must be considered both in crystallograp hic refinement and in analysis of binding mechanisms. (C) Current Biol ogy Ltd