Changes in the Factor VIII C2 domain upon membrane binding determined by hydrogen-deuterium exchange MS

Biochem J. 2014 Aug 1;461(3):443-51. doi: 10.1042/BJ20140121.

Abstract

Factor VIII enhances the catalytic activity of Factor IXa in a membrane-bound enzyme complex and both proteins are necessary to prevent haemophilia. Tandem lectin-like C domains mediate the membrane binding of Factor VIII and membrane-interactive residues have been identified. However, the available data provide little insight into the dynamic changes that occur upon membrane binding. We used time-based hydrogen-deuterium exchange MS to evaluate the dynamics of FVIII-C2 (Factor VIII C2 domain) alone and when membrane bound. The results confirm the participation of previously identified membrane-interactive loops in the binding mechanism. In addition, they indicate that a long peptide segment, encompassing a membrane-interactive loop and strands of the β-barrel core, is remarkably dynamic prior to membrane binding. The flexibility is reduced following membrane binding. In addition, regions that interact with the A1 and C1 domains have reduced solvent exchange. Thus the isolated C2 domain has extensive flexibility that is subject to stabilization and could be related to interactions between domains as well as between Factor VIII and Factor IXa or Factor X. These results confirm that the proposed membrane-binding loops of the FVIII-C2 interact with the membrane in a manner that leads to protection from solvent exposure.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Chromatography, High Pressure Liquid
  • Deuterium Exchange Measurement
  • Factor VIII / chemistry
  • Factor VIII / genetics
  • Factor VIII / metabolism*
  • Humans
  • Kinetics
  • Mass Spectrometry
  • Models, Molecular*
  • Pepsin A
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Peptide Mapping
  • Phospholipids / chemistry
  • Phospholipids / metabolism*
  • Pliability
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Proteolysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Solubility
  • Surface Properties
  • Unilamellar Liposomes / chemistry
  • Unilamellar Liposomes / metabolism*

Substances

  • Peptide Fragments
  • Phospholipids
  • Recombinant Proteins
  • Unilamellar Liposomes
  • F8 protein, human
  • Factor VIII
  • Pepsin A