title: Residues 110-126 in the A1 domain of factor VIII contain a Ca2+ binding site required for cofactor activity.
abstract: Generation of factor VIII cofactor activity requires divalent metal ions such as Ca2+ or Mn2+. Evaluation of cofactor reconstitution from isolated factor VIIIa subunits revealed the presence of a functional Ca2+ binding site within the A1 subunit. Isothermal titration calorimetry demonstrated at least two Ca2+ binding sites of similar affinity (K(d) = 0.74 microm) within the A1 subunit. Mutagenesis of an acidic residue-rich region in the A1 domain (residues 110-126) homologous to a putative Ca2+ binding site in factor V (Zeibdawi, A. R., and Pryzdial, E. L. (2001) J. Biol. Chem. 276, 19929-19936) and expression of B-domainless factor VIII molecules yielded reagents to probe Ca2+ and Mn2+ binding in a functional assay. Basal activity observed for wild type factor VIII in a metal ion-free buffer was enhanced approximately 2-fold with saturating Ca2+ or Mn2+ and yielded functional K(d) values of 1.2 and 1.40 microm, respectively. Ca2+ binding affinity was greatly reduced (or lost) in several mutants including E110A, E110D, D116A, E122A, D125A, and D126A. Alternatively, E113A, D115A, and E124A showed wild type-like activity with little or no reduction in Ca2+ affinity. However, Mn2+ affinity was minimally altered except for mutant D125A (and D116A). These results are consistent with region 110-126 serving a critical role for Ca2+ coordination with selected residues capable of contributing to a partially overlapping site for Mn2+, and that occupancy of either site is required for maximal cofactor activity.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Hironao
authors.initials: H
authors.lastname: Wakabayashi
authors.forename: Jan
authors.initials: J
authors.lastname: Freas
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005169:Factor VIII / Q000737:chemistry* / Q000235:genetics; D015944:Factor VIIIa / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D007477:Ions; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
chemical_list: D002614:Chelating Agents; D007477:Ions; D008345:Manganese; D015944:Factor VIIIa; D005169:Factor VIII; D004492:Edetic Acid; D015951:Factor Xa; D002118:Calcium