Soluble phosphatidylserine binds to a single identified site in the C2 domain of human factor Va.
Factor V(a) (FV(a)) is a cofactor for the serine protease factor X(a) that activates prothrombin to thrombin in the presence of Ca(2+) and a membrane surface. FV(a) is a heterodimer composed of one heavy chain (A1 and A2 domains) and one light chain (A3, C1, and C2 domains). We use fluorescence, circular dichroism, and equilibrium dialysis to demonstrate that (1) the FV C2 domain expressed in Sf9 cells binds one molecule of C6PS with a k(d) of approximately 2 microM, (2) stabilizing changes occur in the FV C2 domain upon C6PS binding, (3) the C6PS binding site in the FV C2 domain is located near residue Cys(2113), which reacts with DTNB, and (4) binding to a PS-containing membrane is an order of magnitude tighter than that to soluble C6PS. Coupled with a recently published crystal structure of the C2 domain, these results support a model for the mechanism of C2-membrane interaction.
Srivastava
A
A
Department of Biochemistry and Biophysics, Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
Quinn-Allen
M A
MA
Kim
S W
SW
Kane
W H
WH
Lentz
B R
BR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D015943:Factor Va / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000138:chemical synthesis; D006358:Hot Temperature; D006801:Humans; D008823:Micelles; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012995:Solubility; D018411:Spodoptera / Q000235:genetics; D014162:Transfection
D008823:Micelles; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D015943:Factor Va; C051388:1,2-dioleoyl-sn-glycero-3-phosphoglycerol; C042511:1,2-dioleoylphosphatidylserine; C017251:1,2-oleoylphosphatidylcholine