Functional and structural characterization of factor Xa dimer in solution.
Previous studies showed that binding of water-soluble phosphatidylserine (C6PS) to bovine factor Xa (FXa) leads to Ca2+-dependent dimerization in solution. We report the effects of Ca2+, C6PS, and dimerization on the activity and structure of human and bovine FXa. Both human and bovine dimers are 10(6)- to 10(7)-fold less active toward prothrombin than the monomer, with the decrease being attributed mainly to a substantial decrease in k(cat). Dimerization appears not to block the active site, since amidolytic activity toward a synthetic substrate is largely unaffected. Circular dichroism reveals a substantial change in tertiary or quaternary structure with a concomitant decrease in alpha-helix upon dimerization. Mass spectrometry identifies a lysine (K(270)) in the catalytic domain that appears to be buried at the dimer interface and is part of a synthetic peptide sequence reported to interfere with factor Va (FVa) binding. C6PS binding exposes K(351) (part of a reported FVa binding region), K(242) (adjacent to the catalytic triad), and K(420) (part of a substrate exosite). We interpret our results to mean that C6PS-induced dimerization produces substantial conformational changes or domain rearrangements such that structural data on PS-activated FXa is required to understand the structure of the FXa dimer or the FXa-FVa complex.
Chattopadhyay
Rima
R
Department of Biochemistry and Biophysics and Program in Molecular and Cellular Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA.
Iacob
Roxana
R
Sen
Shalmali
S
Majumder
Rinku
R
Tomer
Kenneth B
KB
Lentz
Barry R
BR
D000107:Acetylation; D000818:Animals; D001777:Blood Coagulation; D002118:Calcium / Q000494:pharmacology; D020134:Catalytic Domain; D002417:Cattle; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D015951:Factor Xa / Q000737:chemistry* / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D011516:Prothrombin / Q000378:metabolism; D012995:Solubility; D012996:Solutions; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D065427:Factor Xa Inhibitors; D009842:Oligopeptides; D010718:Phosphatidylserines; D012996:Solutions; D011516:Prothrombin; D015951:Factor Xa; D008239:Lysine; D002118:Calcium