title: Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation.
abstract: The zymogen prothrombin is composed of fragment 1 containing a Gla domain and kringle-1, fragment 2 containing kringle-2, and a protease domain containing A and B chains. The prothrombinase complex assembled on the surface of platelets converts prothrombin to thrombin by cleaving at Arg-271 and Arg-320. The three-dimensional architecture of prothrombin and the molecular basis of its activation remain elusive. Here we report the first x-ray crystal structure of prothrombin as a Gla-domainless construct carrying an Ala replacement of the catalytic Ser-525. Prothrombin features a conformation 80 Å long, with fragment 1 positioned at a 36° angle relative to the main axis of fragment 2 coaxial to the protease domain. High flexibility of the linker connecting the two kringles suggests multiple arrangements for kringle-1 relative to the rest of the prothrombin molecule. Luminescence resonance energy transfer measurements detect two distinct conformations of prothrombin in solution, in a 3:2 ratio, with the distance between the two kringles either fully extended (54 ± 2 Å) or partially collapsed (≤34 Å) as seen in the crystal structure. A molecular mechanism of prothrombin activation emerges from the structure. Of the two sites of cleavage, Arg-271 is located in a disordered region connecting kringle-2 to the A chain, but Arg-320 is well defined within the activation domain and is not accessible to proteolysis in solution. Burial of Arg-320 prevents prothrombin autoactivation and directs prothrombinase to cleave at Arg-271 first. Reversal of the local electrostatic potential then redirects prothrombinase toward Arg-320, leading to thrombin generation via the prethrombin-2 intermediate.
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA.
authors.forename: Nicola
authors.initials: N
authors.lastname: Pozzi
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Chen
authors.forename: David W
authors.initials: DW
authors.lastname: Gohara
authors.forename: Weiling
authors.initials: W
authors.lastname: Niu
authors.forename: Tomasz
authors.initials: T
authors.lastname: Heyduk
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D018360:Crystallography, X-Ray; D004735:Energy Transfer; D008958:Models, Molecular; D011487:Protein Conformation; D011516:Prothrombin / Q000737:chemistry*; D055672:Static Electricity
chemical_list: D011516:Prothrombin
keywords: Coagulation factors; Enzyme mutation; Enzyme structure; Structural biology; Thrombin; Zymogen