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M509971200v1
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Papers In Press, published online ahead of print June 6, 2006
J. Biol. Chem, 10.1074/jbc.M509971200
Submitted on September 12, 2005
Revised on June 1, 2006
Accepted on June 6, 2006

High resolution structures of p-aminobenzamidine- and benzamidine-VIIa/soluble tissue factor: Unpredicted conformation of the 192-193 peptide bond and mapping of Ca2+, Mg2+, Na+ and Zn2+ sites in factor VIIa

S. Paul Bajaj, Amy E. Schmidt, Sayeh Agah, Madhu S. Bajaj, and Kaillathe Padmanabhan

Orthopaedic Surgery/Mol Biol Inst, UCLA, Los Angeles, CA 90095

Corresponding Author: pbajaj {at}mednet.ucla.edu

Factor VIIa (FVIIa) consists of a gamma-carboxyglutamic acid (Gla) domain, two EGF-like domains and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+-sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Further, FVIIa contains a Na+ and two Zn2+-sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+ and Zn2+. The crystal diffracted to 1.8 Å resolution and the final structure has an R-factor of 19.8%. In this structure, Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+-site, and the protease domain contains one Ca2+, one Na+, and two Zn2+-sites. Ca2+-binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 support the crystal data. Further, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87 Å resolution. However, soaking benzamidine-FVIIa/sTF crystals with D-Phe-Pro-Arg-chloromethylketone results in benzamidine displacement, D-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.


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