Effect of Membrane Binding Upon Intramolecular Dynamics of the Factor VIII C2 Domain.

Abstract 3168

Poster Board III-109

Factor VIII functions as a membrane-bound cofactor in the Factor Xase complex. Binding to a phospholipid membrane enhances activity of the factor VIIIa-factor IXa complex approx. 100,000-fold. While membrane binding increases the net affinities of factor VIIIa for factor IXa and factor X, the major effect of membrane binding is upon the catalytic activity of the assembled complex. The mechanism through which activity is enhanced remains largely unknown. The C2 domain of factor VIII contains the major membrane-binding function. The x-ray crystal structures of the C2 domain and, subsequently, of intact factor VIII have enabled hypotheses about the mechanism of membrane binding and the enhancement of activity. Identified functional motifs of the C2 domain include two pairs of hydrophobic, membrane-interactive amino acids at the tips of “spikes,” on the lower end of the C2 domain and a putative factor IXa-binding peptide on an upper surface.

Hydrogen/Deuterium Exchange Mass Spectrometry (DXMS) has proven to be an effective method for characterizing protein conformational change induced by ligand interaction. Amide backbone hydrogens of a protein readily exchange with those in the solvent, contingent upon physical contact of the solvent hydrogen with the amide hydrogen. Therefore the exchange rate in a particular region of the protein is dependent on amide hydrogen solvent accessibility. DXMS is performed by isotopic labeling of a protein at various time points with deuterium oxide (D₂O) followed by quenching the exchange and proteolysis to produce overlapping peptides spanning the length of the sequence. The degree of labeling is assessed using liquid chromatography coupled with mass spectrometry analysis. Using this analysis a stability map of the protein can be determined that represents changes in global and local structural dynamics in bound and unbound conditions.

We have utilized DXMS to characterize the dynamics of the factor VIII C2 domain (fVIII-C2) in solution and when bound to phospholipid vesicles. fVIII-C2 was produced in E. coli and purified by immobilized metal affinity chromatography and ion exchange chromatography. Preliminary studies indicated that digestion on serial pepsin and V8 protease columns produced peptides spanning the entire fVIII-C2 structure, with overlapping peptides spanning the membrane-interactive “spikes” and the putative factor IXa-interactive peptide. Exchange was rapid on the membrane-interactive spikes in the absence of phospholipid vesicles, confirming flexibility of these structures. Upon binding of fVIII-C2 to phospholipid vesicles, the exchange rate decreased particularly in spike 3 (from 73% to 49% at 10s labeling, upon addition of phospholipid vesicles). The extent of the region with slowed exchange implied increased structural rigidity in addition to any solvent protection that occurs upon immersion of the hydrophobic side chains into the membrane. A marked decrease in exchange rate was also observed in the putative FIXa binding peptide (from 85% to 21% at 10s labeling, upon addition of phospholipid vesicles), while the exchange rates of most of the peptides from the core beta-barrel of fVIII-C2 were not significantly affected. These observations indicate that membrane engagement alters the mobility of membrane interactive spikes and suggest that membrane binding has an allosteric effect on the putative factor IXa binding site. Membrane-induced allosteric changes in the factor VIII C2 domain may contribute to the major enhancement of factor VIIIa-factor IXa activity that occurs upon membrane binding.