CONFORMATIONAL STABILITY OF A THROMBIN-BINDING PEPTIDE DERIVED FROM THE HIRUDIN C-TERMINUS

The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE spectroscopy [Ni, F., Konishi, Y., & Scheraga, H. A. (1990) Biochemistry 29, 4479-4489]. The stability of the thrombin-bound conformation has been characterized further by a combined NMR and theoretical analysis of the conformational ensemble accessible by the hirudin peptide. Medium- and long-range NOE's were found for the free hirudin peptide in aqueous solution and in a mixture of dimethyl sulfoxide and water at both ambient (25-degrees-C) and low (0-degrees-C) temperatures, suggesting that ordered conformations are highly populated in solution. The global folding of these conformations is similar to that in the thrombin-bound state, as indicated by NOE's involving the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Residues Glu(61), Glu(62), Tyr(63), and Leu(64) all contain approximately 50% of helical conformations calculated from the ratio of the sequential d(NN) and d(alpha-N) NOE's. Among the helical ensemble, active 3(10)-helical conformations were found by an analysis of the medium-range [(i,i + 2) and (i,i + 3)] NOE's involving the last six residues of the peptide. An analysis of the side-chain rotamers revealed that, upon binding to thrombin, there may be a rotation around the alpha-CH-beta-CH bond of Ile(59) such that Ile(59) adopts a gauche- (chi-1 = +60) conformation in contrast to the highly populated trans (chi-1 = -60) found for Ile(59) in the free peptide. However, the thrombin-bound conformation of the hirudin peptide is still an intrinsically stable conformer, and the preferred conformational ensemble of the peptide contains a large population of the active conformation. The apparent preference for a gauche- (chi-1 = + 60) side-chain conformation of Ile(59) in the bound state may be explained by the existence of a positively charged arginine residue among the hydrophobic residues in the thrombin exosite.