4HZH

Structure of recombinant Gla-domainless prothrombin mutant S525A

Summary for 4HZH

• Entry DOI: 10.2210/pdb4hzh/pdb
• Related: 3NXP 4HZQ
• Descriptor: Prothrombin, 2-acetamido-2-deoxy-beta-D-glucopyranose (2 entities in total)
• Functional Keywords: prothrombin, kringle, serine protease, coagulation, hydrolase
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 2
• Total formula weight: 121115.60

Authors

Pozzi, N.,Niu, W.,Gohara, D.W.,Chen, Z.,Di Cera, E. (deposition date: 2012-11-15, release date: 2013-06-26, Last modification date: 2024-11-06)

Primary citation

Pozzi, N.,Chen, Z.,Gohara, D.W.,Niu, W.,Heyduk, T.,Di Cera, E.
Crystal structure of prothrombin reveals conformational flexibility and mechanism of activation.
J.Biol.Chem., 288:22734-22744, 2013

PubMed 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.

PubMed: 23775088
DOI: 10.1074/jbc.M113.466946

Experimental method

X-RAY DIFFRACTION (3.3 Å)