Factor VIII A3 domain residues 1954-1961 represent an A1 domain-interactive site.
Factor VIIIa consists of subunits designated A1, A2, and A3C1C2. Reassociation of the A1 and A3C1C2 subunits monitored by the factor Xa generation assay and fluorescence resonance energy transfer yielded intersubunit affinity values (K(d)) of 58.0 +/- 12.5 and 58.8 +/- 16.8 nM, respectively. These affinity values were equivalent to that previously determined for factor VIII heavy and light chains [Wakabayashi, H., et al. (2001) Biochemistry 40, 10293-10300], suggesting that the A2 domain makes a minimal contribution to the interchain affinity in factor VIII. Ca(2+) showed no effect on A1/A3C1C2 intersubunit affinity (K(d) = 51.6 +/- 16.6 nM), while Cu(2+) enhanced the A1/A3C1C2 intersubunit affinity approximately 5-fold (K(d) = 12.5 +/- 2.3 nM). A synthetic peptide to A3 domain residues 1954-1961 inhibited association of the A1 and A3C1C2 subunits (K(i) = 65.8 +/- 11.9 microM). Three factor VIII point mutants, His1957Ala, Gly1960Val, and His1961Asp, were stably expressed in BHK cells and purified. All mutants exhibited reduced specific activity (39, 42, and 4%, respectively) compared with that of wild-type factor VIII, and their activity was less stable following heat denaturation analysis (t(1/2) values of 13.3 +/- 0.7, 8.7 +/- 0.3, and 8.1 +/- 0.1 min, respectively) compared with that of the wild type (18.8 +/- 0.8 min). This reduced stability appeared to result from an approximately 2-fold increased dissociation rate for the Gly1960Val and His1961Asp dimers as judged by solid-phase binding assays. We propose that residues 1954-1961 of the A3 domain contribute to interactions with the A1 domain, facilitating their association in factor VIII.
Ansong
Charles
C
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Fay
Philip J
PJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D003300:Copper / Q000494:pharmacology; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D002413:Cations, Divalent; D010446:Peptide Fragments; D021122:Protein Subunits; D003300:Copper; D005169:Factor VIII; D002118:Calcium