Thrombin-bound conformation of the C-terminal fragments of hirudin determined by transferred nuclear Overhauser effects

Abstract

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 .alpha.CH proton of Glu(61) and the NH proton of Leu(64)[d.alpha.N(i,ti+3)], between the .alpha.CH proton of Glu(61) and the .beta.CH2 protons of LEu(64) [d.alpha..beta.(i,i+3)], and between the .alpha.CH proton of GLu(62) and the .GAMMA.CH2 protons of GLn(65) [D.alpha..gamma.(I,i+3)]. These NOEs are characteristic of an .alpha.-helical structure involving residues GLu(61) to Gln(65). There are also NOEs between the side-chain protons of residues Phe(56), Ile(599), PRo(60), Tyr(63) and Leu(64). Distance geometry calculations suggest that in the structure of the thrmbin-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).