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 ϳ 40 nM), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K d ‫؍‬ 3-38 M). 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.Factor XI (FXI), 3 the zymogen form of a serine protease coagulation enzyme that is essential for normal hemostasis, is activated either by FXIIa or by thrombin or by autoactivation (1, 2). Once converted to FXIa, the enzyme recognizes its natural macromolecular substrate, FIX, the Ca 2ϩ -dependent activation of which requires the exposure of a substrate-binding site within the Apple 2 (A2) and/or Apple 3 (A3) domains of FXIa and the ␥-carboxyglutamic acid domain of FIX, as well as an extended, macromolecular substrate-binding exosite in the protease domain of FXIa (3-9). The activation of FIX to FIXa␤ involves two cleavages by FXIa, one after Arg-145 and the other after Arg-180, thereby releasing an 11-kDa activation peptide (3,4,10). FIX is also activated to FIXa␤ by the tissue factorFVIIa complex (11).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). The high homology between the heavy chain of FXI and PK indicates a common origin of these two zymogens, in contrast to FXII and other coagulation factors (13,14). However, FXI is a homodimer of two identical subunits joined by a disu...

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.

Yeast Cleavage Factor I (CF I) is an essential complex of five proteins that binds signal sequences at the 3′ end of yeast mRNA. CF I is required for correct positioning of a larger protein complex, CPF, which contains the catalytic subunits executing mRNA cleavage and polyadenylation. CF I is composed of two parts, CF IA and Hrp1. The CF IA has only four subunits, Rna14, Rna15, Pcf11, and Clp1, but the structural organization has not been fully established. Using biochemical and biophysical methods, we demonstrate that CF IA can be reconstituted from bacterially-expressed proteins and that it has 2:2:1:1 stoichiometry of its four proteins, respectively. We also describe mutations that disrupt the dimer interface of Rna14 while preserving the other subunit interactions. Based on our results and existing interaction data, we present a topological model for heterohexameric CF IA and its association with RNA and Hrp1.

It has been known that factor XIa (XIa) can react covalently with antithrombin III (ATIII) to form two complexes with ratios of 2ATIII:1XIa and 1ATIII:1XIa. In the hands of these investigators, the M.W.s, as measured by SDS-PAGE, were 265 kDa and 225 kDa, respectively. In this investigation it has been observed that the addition of 1 µg or 5 µg heparin (H) to 3.1 ϫ 10-5 (mol ATIII for 5 minutes prior to the addition of 1.125 µ 10-5 µmol XIa for a 30-minute incubation led to an increase in the 265 kDa band of 113% and 223%, respectively. These results were statistically significant (p < .01). However, when H was premixed with XIa first, before the addition of AT III, statistical increases in the 265 kDa band were also seen (267% and 183%, respectively; p < .0005, n = 6). Protamine sulfate (PS) statistically significantly (p < .05) inhibited the formation of the 265 and 225 kDa XIa-ATIII complexes at the 5 µg PS level when premixed with ATIII or XIa, respectively, the decreases in the 265 kDa band being 39.1 and 34.4%, respectively for [(ATIII/PS) + XIA] and [(XIa/PS) + ATIII] mixtures, and 23.1 and 23.8% for the 225 kDa band with [(ATIII/PS) + XIa] and [(XIa/PS) + ATIII] mixtures. These results with PS indicate that PS inhibits complex formation between XIa and ATIII at the 2ATIII:1 XIa and 1ATIII:1 XIa levels, in contrast with its reported stimulation of complex formation between thrombin and ATIII.

It has been known that factor XIa (XIa) can react covalently with antithrombin III (ATIII) to form two complexes with ratios of 2ATIII:1XIa and 1ATIII:1XIa. In the hands of these investigators, the M.W.s, as measured by SDS-PAGE, were 265 kDa and 225 kDa, respectively. In this investigation it has been observed that the addition of 1 μg or 5 μg heparin (H) to 3.1 × 10-5 (mol ATIII for 5 minutes prior to the addition of 1.125 μ 10-5 μmol XIa for a 30-minute incubation led to an increase in the 265 kDa band of 113% and 223%, respectively. These results were statistically significant (p < .01). However, when H was premixed with XIa first, before the addition of AT III, statistical increases in the 265 kDa band were also seen (267% and 183%, respectively; p < .0005, n = 6). Protamine sulfate (PS) statistically significantly (p < .05) inhibited the formation of the 265 and 225 kDa XIa-ATIII complexes at the 5 μg PS level when premixed with ATIII or XIa, respectively, the decreases in the 265 kDa band being 39.1 and 34.4%, respectively for [(ATIII/PS) + XIA] and [(XIa/PS) + ATIII] mixtures, and 23.1 and 23.8% for the 225 kDa band with [(ATIII/PS) + XIa] and [(XIa/PS) + ATIII] mixtures. These results with PS indicate that PS inhibits complex formation between XIa and ATIII at the 2ATIII:1 XIa and 1ATIII:1 XIa levels, in contrast with its reported stimulation of complex formation between thrombin and ATIII.