HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 34, 24873-24888, August 25, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/34/24873    most recent M509971200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bajaj, S. P. Articles by Padmanabhan, K. Search for Related Content PubMed PubMed Citation Articles by Bajaj, S. P. Articles by Padmanabhan, K. Social Bookmarking                      What's this?

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 Bajaj¹, Amy E. Schmidt², Sayeh Agah, Madhu S. Bajaj, and Kaillathe Padmanabhan^¶

From the Protein Science Laboratory, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, and the Department of Medicine-Pulmonary Division, UCLA, Los Angeles, California 90095 and the ^¶Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Factor VIIa (FVIIa) consists of a -carboxyglutamic acid (Gla) domain, two epidermal growth factor-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. Furthermore, 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 1.8Å resolution, and the final structure has an R-factor of 19.8%. In this structure, the 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. ⁴⁵Ca²⁺ binding in the presence/absence of Mg²⁺ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly¹⁹³ in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87Å resolution. However, soaking benzamidine-FVIIa/sTF crystals with D-Phe-Pro-Arg-chloromethyl ketone 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 the 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.

Received for publication, September 12, 2005 , and in revised form, June 1, 2006.

The atomic coordinates and structure factors (code 2A2Q, 2AER, and 2FIR) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by National Institutes of Health Grants HL-70369 and HL-36365. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

² Supported in part by a fellowship from the American Association of University Women Educational Foundation.

¹ To whom correspondence should be addressed: UCLA/Orthopaedic Hospital, Dept. of Orthopaedic Surgery, Molecular Biology Institute, Box 951795, Rehab 22-53, Los Angeles, CA 90095-1795. Tel.: 310-825-5622; Fax: 310-825-5972; E-mail: pbajaj{at}mednet.ucla.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A. Hacisalihoglu, P. Panizzi, P. E. Bock, R. M. Camire, and S. Krishnaswamy Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase J. Biol. Chem., November 9, 2007; 282(45): 32974 - 32982. [Abstract] [Full Text] [PDF]

				H. H. Versteeg and W. Ruf Tissue Factor Coagulant Function Is Enhanced by Protein-disulfide Isomerase Independent of Oxidoreductase Activity J. Biol. Chem., August 31, 2007; 282(35): 25416 - 25424. [Abstract] [Full Text] [PDF]

				F. Marino, Z.-W. Chen, C. E. Ergenekan, L. A. Bush-Pelc, F. S. Mathews, and E. Di Cera Structural Basis of Na+ Activation Mimicry in Murine Thrombin J. Biol. Chem., June 1, 2007; 282(22): 16355 - 16361. [Abstract] [Full Text] [PDF]

				M. Ndonwi, G. J. Broze Jr., S. Agah, A. E. Schmidt, and S. P. Bajaj Substitution of the Gla Domain in Factor X with That of Protein C Impairs Its Interaction with Factor VIIa/Tissue Factor: LACK OF COMPARABLE EFFECT BY SIMILAR SUBSTITUTION IN FACTOR IX J. Biol. Chem., May 25, 2007; 282(21): 15632 - 15644. [Abstract] [Full Text] [PDF]

				O. H. Olsen, K. D. Rand, H. Ostergaard, and E. Persson A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation Protein Sci., April 1, 2007; 16(4): 671 - 682. [Abstract] [Full Text] [PDF]