title: Factor XI homodimer structure is essential for normal proteolytic activation by factor XIIa, thrombin, and factor XIa.
abstract: Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXI(C321S) is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXI(C321S), FXI(C321S,K331A), FXI(C321S,E287A), FXI(C321S,I290A), FXI(C321S,Y329A), FXI(C321S,L284A), FXI(C321S,K331R), and FXI(C321S,H343A) were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXI(C321S) and FXI(C321S,H343A) existed in monomer/dimer equilibrium (K(d) approximately 40 nm), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K(d)=3-38 microm). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.
authors.affiliation: Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
authors.forename: Wenman
authors.initials: W
authors.lastname: Wu
authors.forename: Dipali
authors.initials: D
authors.lastname: Sinha
authors.forename: Sergei
authors.initials: S
authors.lastname: Shikov
authors.forename: Calvin K
authors.initials: CK
authors.lastname: Yip
authors.forename: Thomas
authors.initials: T
authors.lastname: Walz
authors.forename: Paul C
authors.initials: PC
authors.lastname: Billings
authors.forename: James D
authors.initials: JD
authors.lastname: Lear
authors.forename: Peter N
authors.initials: PN
authors.lastname: Walsh
mesh_terms: D019943:Amino Acid Substitution; D018360:Crystallography, X-Ray; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D005172:Factor XI / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015956:Factor XIIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015945:Factor XIa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D005172:Factor XI; D015945:Factor XIa; D015956:Factor XIIa; D013917:Thrombin