THROMBIN-BOUND CONFORMATION OF THE C-TERMINAL FRAGMENTS OF HIRUDIN DETERMINED BY TRANSFERRED NUCLEAR OVERHAUSER EFFECTS

The interaction of the C-terminal fragments (residues 52–65 and 55–65) of the thrombin-specific inhibitor hirudin with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution. Thrombin induces specific line broadening of the proton resonances of residues Asp(55) to Gln(65) of the synthetic hirudin fragments H-Asn-Asp-Gly-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH and acetyl-Asp(55)-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(63)-Leu-Gln-COOH. This demonstrates that residues 55–65 are the predominant binding site of hirudin fragments with thrombin. Hirudin fragments take on a well-defined structure when bound to thrombin as indicated by several long-range transferred NOEs between the backbone and side-chain protons of the peptides, but they are not structured when free in solution. Particularly, transferred NOEs exist between the αCH proton of Glu(61) and the NH proton of Leu(64) [dαN(i,i+3)], between the αCH proton of Glu(61) and the ßCH2 protons of Leu(64) [dαß(i,i+3)], and between the aCH proton of Glu(62) and the γCH2 protons of Gln(65) [dαγ(i,i+3)]. These NOEs are characteristic of an α-helical structure involving residues Glu(61) to Gln(65). There are also NOEs between the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Distance geometry calculations suggest that in the structure of the thrombin-bound hirudin peptides all the charged residues lie on the opposite side of a hydrophobic cluster formed by the nonpolar side chains of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). © 1990, American Chemical Society. All rights reserved.