Factor VIII A1 domain residues 97-105 represent a light chain-interactive site.
Results from a recent study on subunit association in factor VIIIa indicated that the A1 and A3C1C2 domains contribute approximately 90% of the interchain binding energy in factor VIII and that A3 domain residues 1954-1961 participate in the interaction with A1 domain (Ansong, C., and Fay, P. J. (2005) Biochemistry 44, 8850-8857). Enhanced trypsin-accessibility at four sites within residues 89-142 in free A1 compared with that of A3C1C2-bound A1, as determined by mass spectrometry, suggested that residues within this region are interactive with the A3C1C2 domains. A synthetic peptide to A1 domain residues 97-105, predicted to be A3 domain-interactive from molecular modeling, inhibited the formation of a functional A1/A3C1C2 dimer (apparent K(i) = 0.64 +/- 0.21 microM) and reduced the efficiency of energy transfer between a fluorescein-labeled A1 subunit and an acrylodan-labeled A3C1C2 subunit. B-domainless factor VIII point mutants, His99Ala, Val101Ala, and Gly102Ser, exhibited reduced specific activity (32%, 51%, and 45%, respectively) compared with that of factor VIII wild type. Furthermore, the activity of factor VIII His99Ala was less stable (t(1/2) = 2.3 +/- 0.2 min) compared with that of factor VIII wild type (t(1/)(2) = 6.2 +/- 0.7 min) following heat denaturation analysis. This reduced stability appeared to result from an approximately 40% increase in the dissociation rate for the mutant factor VIII heterodimer as judged by solid-phase binding assays. These results indicate that residues within segment 97-105 of the A1 domain interact with residues within the A3C1C2 domains of the light chain and contribute to the interface in the factor VIII heterodimer.
Ansong
Charles
C
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642, USA.
Miles
Stephen M
SM
Fay
Philip J
PJ
D000595:Amino Acid Sequence; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D005169:Factor VIII