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J. Biol. Chem., Vol. 277, Issue 3, 1855-1863, January 18, 2002

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Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor X_a^*

Arvind Srivastava, Jianfang Wang, Rinku Majumder, Alireza R. Rezaie^§, Johan Stenflo^¶, Charles T. Esmon, and Barry R. Lentz^**

From the  Department of Biochemistry & Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, the ^¶ Department of Clinical Chemistry, University of Lund, Malmö General Hospital, Malmö S214 01, Sweden, the ^§ Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, and the  Department of Biochemistry and Biophysics and Pathology, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center and Howard Hughes Medical Institute, Oklahoma City, Oklahoma 73104

Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X_a by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X_a using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X_a. Our results demonstrate that the structural responses of human and bovine factor X_a to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X_a are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the -carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca²⁺ (k_d ~ 1 mM), although this PS site does not influence the functional response of factor X_a. Second, a Ca²⁺-dependent binding site is in the epidermal growth factor domains (EGF_NC) that are linked by Ca²⁺ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X_a. Third, a Ca²⁺-requiring site seems to be in the EGF_C-catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF_NC, and catalytic domains in the presence of Ca²⁺, meaning that PS regulation of factor X_a involves linkage between widely separated parts of the protein.

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