Crystal Structures of the FXIa Catalytic Domain in Complex with Ecotin Mutants Reveal Substrate-like Interactions

Abstract

Thrombosis can lead to life-threatening conditions such as acute myocardial infarction, pulmonary embolism, and stroke. Although commonly used anti-coagulant drugs, such as low molecular weight heparin and warfarin, are effective, they carry a significant risk of inducing severe bleeding complications, and there is a need for safer drugs. Activated Factor XI (FXIa) is a key enzyme in the amplification phase of the coagulation cascade. Anti-human FXI antibody significantly reduces thrombus growth in a baboon thrombosis model without bleeding problems (Gruber, A., and Hanson, S. R. (2003) Blood 102, 953-955). Therefore, FXIa is a potential target for anti-thrombosis therapy. To determine the structure of FXIa, we derived a recombinant catalytic domain of FXI, consisting of residues 370 -607 (rhFXI 370 -607 ). Here we report the first crystal structure of rhFXI 370 -607 in complex with a substitution mutant of ecotin, a panserine protease protein inhibitor secreted by Escherichia coli, to 2.2 Å resolution. The presence of ecotin not only assisted in the crystallization of the enzyme but also revealed unique structural features in the active site of FXIa. Subsequently, the sequence from P5 to P2 in ecotin was mutated to the FXIa substrate sequence, and the structures of the rhFXI 370 -607 -ecotin mutant complexes were determined. These structures provide us with an understanding of substrate binding interactions of FXIa, the structural information essential for the structure-based design of FXIa-selective inhibitors.

Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2

“…The protein was cloned, expressed, and purified as described previously. Throughout this paper, the chymotrypsin sequence numbering system is used for FXIac in order to be consistent with other published data on trypsin-like serine proteases (see supplemental material in Jin et al (29) for the corresponding residue numbers for the FXI sequence and chymotrypsin numbering system) 5 .…”

“…For ease of discussion, recombinant human FXI-(370 -607)-S434A,T475A,C482S will be termed FXIac, whereas the unmutated form will be referred to as FXI-(370 -607), consistent with our previous publication (29). The protein was cloned, expressed, and purified as described previously.…”

“…We have determined the crystal structures of the catalytic domain of FXIa (rhFXI-(370 -607)) with ecotin, an Escherichia coli serine protease inhibitor (29), as well as benzamidine, a small molecule inhibitor of trypsin-like serine proteases. 5 In the present study, we report the crystal structure of a complex of FXIac (FXI-(370 -607)-S434A,T475A,C482S) and PN2KPI.…”

Characterization of seven novel mutations causing factor XI deficiency

“…13 The solvent-accessible area for each residue in both FXI and FXIa structures was calculated using the SURFV program with a probe sphere with a radius of 1.4Å and default parameters. 14 Evolutionary conservation analysis was performed using the ConSurf web-server 15 (http://consurf.tau.ac.il).…”

Solution structure of the A4 domain of factor XI sheds light on the mechanism of zymogen activation

“…Once activated, the only covalent linkage between the heavy chain of FXIa (residues 1-369) and the catalytic domain (residues 370-607) is through a disulfide bond between Cys-362 and Cys-482 (4). The newly generated NH 3 ϩ group of the N-terminal Ile-370 moves over a distance of Ϸ20 Å into close proximity to the catalytic site and interacts electrostatically with active-site residues, including 21). Although this phenomenon has long been known to play a key role in the activation of serine proteases (22), the fate of the newly created C-terminal end of the heavy chain and its influence on the regulatory domains remain to be elucidated.…”