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 dNN 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.
Ni
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F
Protein Engineering Section, National Research Council of Canada, Montréal, Québec.
Ripoll
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DR
Purisima
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EO
D001665:Binding Sites; D002942:Circular Dichroism; D004355:Drug Stability; D006629:Hirudins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012996:Solutions; D013917:Thrombin / Q000378:metabolism*; D014867:Water
D006629:Hirudins; D010446:Peptide Fragments; D012996:Solutions; D014867:Water; D013917:Thrombin