The co-crystal structure of unliganded bovine alpha-thrombin and prethrombin-2: movement of the Tyr-Pro-Pro-Trp segment and active site residues upon ligand binding.
Unliganded bovine alpha-thrombin and prethrombin-2 have been co-crystallized, in space group P21212, using either ammonium sulfate or polyethylene glycol 2000 (PEG2K), and their structures determined at 2.2 A and 2.3 A, respectively. Initial phases were determined by molecular replacement and refined using XPLOR to final R factors of 0.187 (Rfree = 0.255) and 0.190 (Rfree = 0.282) for the salt and PEG2K models, respectively. The apo-enzyme form of bovine alpha-thrombin shows dramatic shifts in placement for the Tyr-Pro-Pro-Trp segment, for Glu-192, and for the catalytic residues His-57 and Ser-195, when compared to 4 thrombin complexes representing different states of catalysis, namely (1) the Michaelis complex (residues 7-19 of fibrinogen A alpha with a non-cleavable scissile bond), (2) enzyme-inhibitor complex (D-Phe-Pro-Arg chloromethylketone), (3) enzyme product complex (residues 7-16 of fibrinopeptide A), and (4) the exosite complex (residues 53-64 of hirudin). The structures of bovine and human prethrombin-2 are generally similar to one another (RMS deviation of 0.68 A) but differ significantly in the Arg-15/Ile-16 cleavage region and in the three activation domains, which are disordered in bovine prethrombin-2, analogous to that seen for trypsinogen.
Malkowski
M G
MG
Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, Michigan 48201, USA.
Martin
P D
PD
Guzik
J C
JC
Edwards
B F
BF
D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D002417:Cattle; D018360:Crystallography, X-Ray; D004792:Enzyme Precursors / Q000737:chemistry*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D009038:Motion; D011392:Proline; D011487:Protein Conformation; D011516:Prothrombin / Q000737:chemistry*; D013045:Species Specificity; D013917:Thrombin / Q000737:chemistry*; D014364:Tryptophan; D014443:Tyrosine
D004792:Enzyme Precursors; D008024:Ligands; D014443:Tyrosine; D014364:Tryptophan; D011516:Prothrombin; D005340:Fibrinogen; C012007:prethrombins; D011392:Proline; D013917:Thrombin