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 alpha CH proton of Glu(61) and the NH proton of Leu(64) [d alpha N(i,i+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(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).
Ni
F
F
Section of Protein Engineering, National Research Council Canada, Montréal, Québec.
Konishi
Y
Y
Scheraga
H A
HA
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006629:Hirudins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*
D006629:Hirudins; D009842:Oligopeptides; D010446:Peptide Fragments; D013917:Thrombin