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.

FIGURE 1.

Structure of FXI dimer interface and characterization of FXI mutants by size exclusion chromatography and analytical ultracentrifugation. A–C, structure of the FXI dimer interface based on the crystal structure of FXI (19) predicting a salt bridge between the positively charged Lys-331 residue of one subunit 2.47 Å away from the negatively charged Glu-287 residue on the opposite subunit (A); a hydrophobic interaction (3.57 Å) between Leu-284 and Ile-290 (B); and a hydrophobic interaction (3.92 Å) between the two Tyr-329 residues of the two A4 domains (C). D and E, size exclusion chromatography elution profiles, carried out as described under the “Experimental Procedures.” The retention volumes of purified plasma FXI and PK, 11.2 and 12.3 ml, were adopted as references for dimer and monomer, respectively (D). The mutant FXI_C321S migrated with only one peak that had a retention volume, 11.5 ml, similar to that of the FXI dimer, whereas the FXI_C321S,K331A mutant displayed a retention volume of 12.4 ml (E), i.e. similar to PK (D). All the other monomeric double mutants also migrated with one predominant peak that had the retention volume around that of PK even at a concentration of 200 μg/ml (data not shown). F–H, equilibrium sedimentation analysis, performed as described under the “Experimental Procedures” at 12,000 rpm (upper curve, red), 15,000 rpm (middle curve, green), and 20,000 rpm (lower curve, blue) for 20–24 h. The radial absorbance profiles (points) and fits (lines) to equations describing equilibrium sedimentation of FXI (F) and PK (G) are shown. The FXI_C321S,K331A mutant was selected as a representative of all the mutants as shown in H. The bottom part of each panel showed the species plot of weight fraction against concentration. For FXI and PK, there was only one species (upper black curves); however, the mutant FXI_C321S,K331A existed in the equilibrium of dimer (upper black curve) and monomer (lower blue curves), and the dimer species became dominant with the increment of concentration.

FIGURE 2.

Electron microscopy of WTFXI, FXI_C321S,K331A, and PK. A, raw image (left) and representative class averages (right, numbered 1–6) of WTFXI, showing the wild-type protein forming a dimer. B, raw image (left) and representative class averages (right, numbered 1–6) of FXI_C321S,K331A, showing that the mutations prevent the protein from dimerizing. C, raw image (left) and representative class averages (right, numbered 1–6) of PK, revealing its structural similarity to FXI_C321S,K331A. The scale bar represents 25 nm, and the side length of the panels showing individual class averages is 22.5 nm.

FIGURE 3.

FXI activation by FXIIa and thrombin and autoactivation. A, activation of FXI and the mutants (30 nm) by FXIIa (3 nm) in solution were carried out in TBSA buffer (NaCl 150 mm, Tris-HCl 50 mm, pH 7.4, 0.1% BSA). The incubation mixtures were sampled at the indicated time points; the activity of FXIIa was inhibited by CTI, and the generation of FXIa was determined by its capacity to cleave the synthetic substrate S2366 (330 μm). B, activation of FXI and the mutants (30 nm) by FXIIa (3 nm) was carried out in the presence of 1 μg/ml of dextran sulfate (500 kDa) in TBSA buffer. C, FXI_C321S,K331A mutant (30 nm) was activated by FXIIa at different enzyme:substrate molar ratios, 1:10 (○), 1:5 (▪), and 1:1 (▾). The reactions were carried out in TBSA buffer (NaCl 150 mm, Tris-HCl 50 mm,pH 7.4, 0.1% BSA). The FXIIa activity was inhibited by CTI as described under “Experimental Procedures,” and the generation of FXIa was determined by its capacity to cleave the synthetic substrate S2366 (330 μm). D, activation of WTFXI and mutants (30 nm) by thrombin (1 nm) was carried out in the presence of 1 μg/ml of dextran sulfate (500 kDa) in TBSA buffer. The thrombin was inhibited by addition of hirudin (5 nm) as described under “Experimental Procedures,” and the generation of FXIa was determined by its capacity to cleave the synthetic substrate S2366 (330 μm). E, autoactivation of FXI and mutants (30 nm) on dextran sulfate (1 μg/ml, 500 kDa) surface in TBSA buffer (NaCl 150 mm, Tris-HCl 50 mm, pH 7.4, 0.1% BSA). The incubation mixtures were sampled at the indicated time points, and the generation of FXIa was determined by its capacity to cleave the synthetic substrate S2366 (330 μm). Data are shown for activation of WTFXI (○), FXI_C321S (▪), FXI_C321S,H343A (▴), and FXI_C321S,K331A (▾). Results (not shown) for the monomeric mutants FXI_C321S,E287A, FXI_C321S,I290A, FXI_C321S,Y329A, and FXI_C321S,L284A were virtually identical to those for FXI_C321S,K331A.

FIGURE 4.

FIX activation by FXIa mutants. A, activation of the monomeric mutant FXI_C321,K331A (200 nm) by FXIIa (100 nm) was carried out in TBS buffer (NaCl 150 mm, Tris-HCl 50 mm, pH 7.4), and the conversion of zymogen to enzyme at different time points was examined by SDS-PAGE under reducing conditions. The conversion of FXI (80 kDa) to FXIa (50 kDa heavy chain, HC, and 30 kDa light chain, LC) was detected by Western blot with anti FXI polyclonal antibody. B and C, activation of FIX (400 nm) by FXIa or activated monomeric mutant (2 nm) was carried out in TBS buffer (NaCl 150 mm, Tris-HCl 50 mm, pH 7.4) in the presence of 2 mm CaCl₂. At the indicated time point samples were removed, and the reaction was stopped by adding 10% SDS buffer containing 10% β-mercaptoethanol. The samples were subjected to SDS-PAGE after boiling for 3 min. Representative data are shown for WTFXIa (B) and the activated mutant FXIa_C321S,K331A (C). Very similar results were obtained with all other mutants, including FXIa_C321S,H343A, FXIa_C321S,E287A, FXIa_C321S/I290A, FXIa_C321S,Y329A, and FXIa_C321S,L284A.