title: Modification of interdomain interfaces within the A3C1C2 subunit of factor VIII affects its stability and activity.
abstract: Factor (F)VIII consists of a heavy chain [A1(a1)A2(a2)B domains] and a light chain [(a3)A3C1C2 domains]. Several reports have shown significant changes in FVIII stability and/or activity following selected mutations at the A1-A2, A1-A3, A2-A3, and A1-C2 domain interfaces. In this study, the remaining inter-FVIII subunit interfaces (A3-C1 and C1-C2) were examined for their contributions to the stability and activity of FVIII and FVIIIa. We prepared FVIII mutants with nascent disulfide bridges between A3 and C1 domains (Gly1750Cys/Arg2116Cys and Ala1866Cys/Ser2119Cys) or C1 and C2 domains (Ser2029Cys/Pro2292Cys). We also prepared mutants via replacement of Arg2116 with hydrophobic residues (Ala and Val) because this C1 domain residue appears to face a pocket of positive electrostatic potential in the A3 domain. Stability was assessed following the rates of loss of FVIII activity at 55 °C and the spontaneous loss of FVIIIa activity from A2 subunit dissociation. FVIII Gly1750Cys/Arg2116Cys showed a marked increase in thermal stability (∼3.7-fold) compared with that of wild-type (WT) FVIII, while the stability of FVIII Ala1866Cys/Ser2119Cys was reduced (∼4.7-fold). Although the Ser2029Cys/Pro2292Cys variant showed a modest loss of FVIII stability, the specific activity and thrombin generation potential of this variant were increased (up to 1.2-fold) compared with those of WT. Furthermore, this variant demonstrated an ∼2-fold reduced Km for FX. Mutation of Arg2116 to hydrophobic residues resulted in variable decreases in stability and thrombin generation parameters, suggesting a role of this Arg residue contributing to FVIII structure. Taken together, selective modification of the contiguous domain interfaces in the FVIII light chain may improve FVIII stability and/or cofactor function.
authors: Department of Biochemistry and Biophysics, University of Rochester School of Medicine , 601 Elmwood Avenue, Rochester, New York 14642, United States.
authors: Hironao
authors: H
authors: Wakabayashi
authors: Philip J
authors: PJ
authors: Fay
mesh_terms: D019943:Amino Acid Substitution; D004220:Disulfides / Q000737:chemistry; D005169:Factor VIII / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015944:Factor VIIIa / Q000737:chemistry; D005170:Factor X / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009154:Mutation; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D055672:Static Electricity
chemical_list: D004220:Disulfides; D015944:Factor VIIIa; D005169:Factor VIII; D005170:Factor X
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