Localization of phosphatidylserine binding sites to structural domains of factor Xa.
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 gamma-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca(2+) (k(d) approximately 1 mm), although this PS site does not influence the functional response of factor X(a). Second, a Ca(2+)-dependent binding site is in the epidermal growth factor domains (EGF(NC)) that are linked by Ca(2+) and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X(a). Third, a Ca(2+)-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 Ca(2+), meaning that PS regulation of factor X(a) involves linkage between widely separated parts of the protein.
Srivastava
Arvind
A
Department of Biochemistry & Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Wang
Jianfang
J
Majumder
Rinku
R
Rezaie
Alireza R
AR
Stenflo
Johan
J
Esmon
Charles T
CT
Lentz
Barry R
BR
D000577:Amides / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010718:Phosphatidylserines / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000577:Amides; D010718:Phosphatidylserines; D015951:Factor Xa