Localization of phosphatidylserine binding sites to structural domains of factor Xa

Abstract

Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor Xa 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 Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor Xa. Our results demonstrate that the structural responses of human and bovine factor Xa to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor Xa are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the γ-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca2+ (kd ∼ 1 mM), although this PS site does not influence the functional response of factor Xa. Second, a Ca2+-dependent binding site is in the epidermal growth factor domains (EGFNC) that are linked by Ca2+ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor Xa. Third, a Ca2+-requiring site seems to be in the EGFC-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, EGFNC, and catalytic domains in the presence of Ca2+, meaning that PS regulation of factor Xa involves linkage between widely separated parts of the protein.