title: Crystallographic structure of human gamma-thrombin.
abstract: In an effort to prepare crystals and determine the structure of alpha-thrombin complexed to a synthetic peptide inhibitor (MDL-28050) of the hirudin 54-65 COOH-terminal region, it was discovered that the crystals were not those of the complex but of gamma-thrombin. Gel electrophoresis studies revealed that autolytic degradation had occurred prior to crystallization. NH2-terminal sequence analysis of these autolytic fragments confirmed the gamma-thrombin product (cleavages at Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150; chymotrypsinogen numbering) with a minor amount of another autolysis product, beta-thrombin (first two cleavages only). The final structure has an R-factor of 0.156 for 7.0-2.5-A data, and includes 186 water molecules. A comparison of gamma-thrombin with the thrombin structure in the alpha-thrombin-hirugen complex revealed that the two structures agreed well (r.m.s. delta = 0.39 A for main chain atoms). These structures possess uninhibited active sites where the disposition of the catalytic triad residues is nearly identical. The electron density in the vicinity of the gamma-thrombin cleavage regions is poor, and only becomes well-defined several residues prior to and after the actual cleavage sites. The extensive disorder evoked by beta-cleavage(s) in the Lys70-Glu80 loop region indicates that this part of the molecule is severely disrupted by autolysis and is the reason exosite functions are dramatically impaired in beta-and gamma-thrombin. Since autolysis did not lead to a major reorganization of the folded structure of alpha-thrombin, the likely structural features of the interaction of thrombin substrate with thrombin enzyme during beta-cleavage have been modeled by docking the exosite region of one molecule at the active site of another.
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing 48824-1322.
authors.forename: T J
authors.initials: TJ
authors.lastname: Rydel
authors.forename: M
authors.initials: M
authors.lastname: Yin
authors.forename: K P
authors.initials: KP
authors.lastname: Padmanabhan
authors.forename: D T
authors.initials: DT
authors.lastname: Blankenship
authors.forename: A D
authors.initials: AD
authors.lastname: Cardin
authors.forename: P E
authors.initials: PE
authors.lastname: Correa
authors.forename: J W
authors.initials: JW
authors.lastname: Fenton
authors.forename: A
authors.initials: A
authors.lastname: Tulinsky
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006629:Hirudins / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D006629:Hirudins; D009842:Oligopeptides; D010446:Peptide Fragments; C062510:hirugen; C064762:MDL 28050; D013917:Thrombin