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Title: Structural architecture of prothrombin in solution revealed by single molecule spectroscopy

The coagulation factor prothrombin has a complex spatial organization of its modular assembly that comprises the N-terminal Gla domain, kringle-1, kringle-2, and the C-terminal protease domain connected by three intervening linkers. Here we use single molecule Förster resonance energy transfer to access the conformational landscape of prothrombin in solution and uncover structural features of functional significance that extend recent x-ray crystallographic analysis. Prothrombin exists in equilibrium between two alternative conformations, open and closed. The closed conformation predominates (70%) and features an unanticipated intramolecular collapse of Tyr 93 in kringle-1 onto Trp 547 in the protease domain that obliterates access to the active site and protects the zymogen from autoproteolytic conversion to thrombin. The open conformation (30%) is more susceptible to chymotrypsin digestion and autoactivation, and features a shape consistent with recent x-ray crystal structures. Small angle x-ray scattering measurements of prothrombin wild type stabilized 70% in the closed conformation and of the mutant Y93A stabilized 80% in the open conformation directly document two envelopes that differ 50 Å in length. These findings reveal important new details on the conformational plasticity of prothrombin in solution and the drastic structural difference between its alternative conformations. Prothrombin uses the intramolecular collapse of kringle-1more » onto the active site in the closed form to prevent autoactivation. As a result, the open-closed equilibrium also defines a new structural framework for the mechanism of activation of prothrombin by prothrombinase.« less
Authors:
 [1] ;  [2] ;  [3] ;  [1]
  1. Saint Louis Univ. School of Medicine, St. Louis, MO (United States)
  2. Saint Louis Univ. School of Medicine, St. Louis, MO (United States); Wroclaw Univ. of Technology, Wroclaw (Poland)
  3. Argonne National Lab. (ANL), Argonne, IL (United States)
Publication Date:
Grant/Contract Number:
AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 35; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; enzyme kinetics; prothrombin; single-molecule biophysics; structure-function; thrombin
OSTI Identifier:
1369035