CRYSTALLOGRAPHIC STRUCTURE OF HUMAN GAMMA-THROMBIN

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 crysta ls were not those of the complex but of gamma-thrombin. Gel electropho resis studies revealed that autolytic degradation had occurred prior t o crystallization. NH2-terminal sequence analysis of these autolytic f ragments confirmed the gamma-thrombin product (cleavages at Arg(75)-Ty r(76) and/or Arg(77A)-Asn(78), and Lys(149E)-Gly(150); chymotrypsinoge n numbering) with a minor amount of another autolysis product, beta-th rombin (first two cleavages only). The final structure has an R-factor of 0.156 for 7.0-2.5-Angstrom data, and includes 186 water molecules. A comparison of gamma-thrombin with the thrombin structure in the alp ha-thrombin-hirugen complex revealed that the two structures agreed we ll (r.m.s.Delta = 0.39 Angstrom for main chain atoms). These structure s possess uninhibited active sites where the disposition of the cataly tic triad residues is nearly identical. The electron density in the vi cinity of the gamma-thrombin cleavage regions is poor, and only become s well defined several residues prior to and after the actual cleavage sites. The extensive disorder evoked by beta-cleavage(s) in the Lys(7 0)-Glu(80) loop region indicates that this part of the molecule is sev erely disrupted by autolysis and is the reason exosite functions are d ramatically impaired in beta- and gamma-thrombin. Since autolysis did not lead to a major reorganization of the folded structure of alpha-th rombin, 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 an other.