title: Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis. Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments.
abstract: Hirudin is the most potent and specific inhibitor of the blood-clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64-amino-acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41-49 of the chain. The N-terminal fragments 1-41 and 1-47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three-dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a N-terminal well-structured domain (core) and a C-terminal flexible polypeptide segment. Determination of the accessible surface area of the three-dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.
authors.affiliation: CRIBI Biotechnology Centre, University of Padua, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Vindigni
authors.forename: V
authors.initials: V
authors.lastname: De Filippis
authors.forename: G
authors.initials: G
authors.lastname: Zanotti
authors.forename: C
authors.initials: C
authors.lastname: Visco
authors.forename: G
authors.initials: G
authors.lastname: Orsini
authors.forename: A
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000378:metabolism*; D006629:Hirudins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D007865:Leeches / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D017433:Protein Structure, Secondary; D012697:Serine Endopeptidases / Q000378:metabolism; D013381:Subtilisins / Q000378:metabolism; D013820:Thermolysin / Q000378:metabolism; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014357:Trypsin / Q000378:metabolism
chemical_list: D004220:Disulfides; D006629:Hirudins; D010446:Peptide Fragments; D010450:Endopeptidases; D012697:Serine Endopeptidases; D013381:Subtilisins; C028344:glutamyl endopeptidase; D014357:Trypsin; D013917:Thrombin; D013820:Thermolysin