Abstract
BackgroundCoagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI.ObjectiveTo investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain.Methods and resultsA series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates.ConclusionsThese data provide the first structural basis for understanding FXII substrate recognition and zymogen activation.
Highlights
FXII is a central component of the contact system, which includes the serine proteinase prekallikrein (PK) and the non-enzymatic cofactor high molecular weight kininogen [1]
It has been recently demonstrated that neutrophil extracellular traps, which form networks of fibers primarily composed of DNA, can activate the contact system in a process linked to innate immunity, the generation of antimicrobial peptides, and complement activation [4]
The FXII Arg353–Val354 peptide bond is cleaved by kallikrein, generating a-FXIIa, which has a heavy chain of 50 kDa, connected to a light chain of 28 kDa by the Cys340–Cys467 disulfide bridge
Summary
FXII is a central component of the contact system, which includes the serine proteinase prekallikrein (PK) and the non-enzymatic cofactor high molecular weight kininogen [1]. The contact system can be activated by diverse negatively charged polymers, including kaolin [1], nucleic acids [2], and collagen [3]. The FXII Arg353–Val354 peptide bond is cleaved by kallikrein, generating a-FXIIa, which has a heavy chain of 50 kDa, connected to a light chain of 28 kDa by the Cys340–Cys467 disulfide bridge. Once a small amount of a-FXIIa is generated, this cleaves PK to generate kallikrein, which mediates efficient cleavage of further FXII in a feedback loop that amplifies production of a-FXIIa and kallikrein. Subsequent cleavage of a-FXIIa results in loss of the heavy chain and generation of the isolated protease domain termed b-FXIIa, which contains only a nine amino acid peptide heavy chain remnant disulfide bonded to the protease domain [1]
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