title: Stabilizing interactions between D666-S1787 and T657-Y1792 at the A2-A3 interface support factor VIIIa stability in the blood clotting pathway.
abstract: Essentials Factor VIIIa (FVIIIa) is unstable due to loss of A2; D666 and Y1792 contribute to its stability. We conducted a study to identify the interactions made at these residues at the A2-A3 interface. We present evidence for stabilizing interactions between D666-S1787 and T657-Y1792 in FVIIIa. A D666C/S1788C variant with a disulfide A2-A3 linkage has a FVIIIa decay rate that is 1% of wild-type.
  
  
  
  Background Factor (F)VIIIa activity and stability depends on the non-covalent association of the A2 subunit with the A1/A3C1C2 dimer, but the interactions that contribute to A2 association are not well defined. Previous work had shown that D666A and Y1792F mutations at the A2-A3 interface resulted in increased FVIIIa decay, suggesting that the residues were involved in bonding interactions important for FVIIIa stability. Objectives Several potential hydrogen bonding partners of D666 and Y1792 across the A2-A3 interface were selected from the low-resolution FVIII crystal structure, and we used mutagenesis and biochemical analysis to examine the bonding interactions occurring at D666 and Y1792. Methods Using a series of stability and functional analyses, we analyzed FVIII variants in which D666 and Y1792 were each swapped with the residues of potential bonding partners. Results and conclusions We present evidence for hydrogen bonds between D666 and S1787 and between Y1792 and T657 that are important for FVIIIa stability. D666S/S1787D and T657Y/Y1792T variants each displayed wild-type (WT)-like FVIIIa stability and performed like WT FVIII in a series of functional analyses, whereas D666S, S1787D, and Y1792T single variants showed increased FVIIIa decay and a T657Y variant had little FVIIIa activity. These results suggest that WT hydrogen bonds are disrupted with the single mutations but maintained in the swap variants. Furthermore, mutation of D666 and S1788 to cysteine resulted in disulfide bond formation across the A2-A3 interface, confirming the close proximity of D666 and S1787, and this covalent attachment of the A2 subunit significantly increased FVIIIa stability.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA.
authors.forename: M
authors.initials: M
authors.lastname: Monaghan
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA.
authors.forename: H
authors.initials: H
authors.lastname: Wakabayashi
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA.|Green Mountain Antibodies, Burlington, VT, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Griffiths
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Fay
mesh_terms: D001777:Blood Coagulation; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides; D005169:Factor VIII / Q000235:genetics; D015944:Factor VIIIa / Q000737:chemistry*; D015951:Factor Xa / Q000737:chemistry; D014644:Genetic Variation; D006467:Hemophilia A / Q000097:blood / Q000276:immunology; D006801:Humans; D006860:Hydrogen Bonding; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D013917:Thrombin / Q000737:chemistry; D013927:Thrombosis / Q000235:genetics
chemical_list: D004220:Disulfides; D015944:Factor VIIIa; D005169:Factor VIII; D013917:Thrombin; D015951:Factor Xa; D003545:Cysteine
keywords: blood coagulation; factor VIII; factor VIIIa; protein stability; site-directed mutagenesis