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.
Highlights
Factor XI (FXI) and plasma prekallikrein (PK) are 58% identical in their amino acid sequences, and the domain structures of the two molecules are very similar, with each molecule containing four homologous apple (A1–A4) domains [12]
FXI is a homodimer of two identical subunits joined by a disulfide bond formed by Cys-321 within the A4 domain of each subunit, whereas PK exists as a monomer [15, 16]
If the interchain disulfide bond formed by Cys-321 in FXI is the only site responsible for homodimer formation, FXIC321S or FXIC321A should be monomeric; both of these mutants exist predominantly as dimers [15, 17], strongly suggesting that other noncovalent interactions are important for maintaining the dimeric structure of FXI
Summary
We aimed to evaluate the relative contributions of selected residues within the A4 domain to dimer formation and to understand the importance of the dimeric structure of FXI to its normal function. After examining the crystal structure of FXI [19] and comparing the amino acid sequence of FXI and PK [12], we were able to predict the candidate residues (Leu-284, Glu-287, Ile-290, Tyr-329, and Lys-331) within the A4 domain that may be involved in homodimer formation. These FXI A4 domain residues were mutated and the mutant proteins expressed in HEK293 cells, and the purified proteins were examined by size exclusion chromatography, analytical ultracentrifugation, and electron microscopy for their capacity to mediate homodimer formation and for their functional properties
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