Interactions outside the proteinase-binding loop contribute significantly to the inhibition of activated coagulation factor XII by its canonical inhibitor from corn

Abstract

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) among other plasma proteases. CHFI is considered a canonical serine protease inhibitor that interacts with FXIIa through its protease-binding loop. Here we examined whether the protease-binding loop alone is sufficient for the selective inhibition of serine proteases or whether other regions of a canonical inhibitor are involved. Six CHFI mutants lacking different N- and C-terminal portions were generated. CHFI-234, which lacks the first and fifth disulfide bonds and 11 and 19 amino acid residues at the N and C termini, respectively, exhibited no significant changes in FXIIa inhibition (Ki = 3.2 ± 0.4 nm). CHFI-123, which lacks 34 amino acid residues at the C terminus and the fourth and fifth disulfide bridges, inhibited FXIIa with a Ki of 116 ± 16 nm. To exclude interactions outside the FXIIa active site, a synthetic cyclic peptide was tested. The peptide contained residues 20-45 (Protein Data Bank code 1BEA), and a C29D substitution was included to avoid unwanted disulfide bond formation between unpaired cysteines. Surprisingly, the isolated protease-binding loop failed to inhibit FXIIa but retained partial inhibition of trypsin (Ki = 11.7 ± 1.2 μm) and activated factor XI (Ki = 94 ± 11 μm). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 nm and activated factor XI with a Ki of 5.4 ± 0.2 μm. Our results suggest that the protease-binding loop is not sufficient for the interaction between FXIIa and CHFI; other regions of the inhibitor also contribute to specific inhibition.

Keywords: Blood Coagulation Factors; Enzyme Kinetics; Enzyme Mechanisms; Protease Inhibitor; Protein Expression; Protein-Protein Interactions; Serine Protease.

FIGURE 1.

CHFI and its mutants. A, schematic diagrams of CHFI mutants. Cysteine residues and terminal amino acids are marked with numbers below each scheme. Numbered square brackets indicate disulfide bridges, triangles indicate the reactive site (i.e. the scissile bond between Arg³⁴ and Leu³⁵), crosses indicate Cys to Asp substitutions, and the ellipse indicates the unpaired cysteine C86. B, schematic diagrams of CHFI mutant conformations. Visualizations were performed using Discovery Studio software (Accelrys, Inc., San Diego, CA). α-Helices are indicated in red, and disulfide bridges are indicated as yellow balls and sticks. Cysteines and Cys to Asp mutations are labeled, and the inhibitory loop is located at the top. C, amino acid sequence alignment of CHFI mutants obtained using ClustalW software (38). The cysteine positions are indicated with blue boxes, and the positions of Cys to Asp mutations are presented in red. Purification tags and amino acids from the expression vector are marked with gray shading. The numbers above the sequence indicate the first and last amino acids from CHFI that are present in the mutants and the Cys residues, according to 1BEA in the PDB. The last number in each sequence line corresponds to the total length of the recombinant protein.

FIGURE 2.

SDS-PAGE analysis of the purity of the recombinant inhibitors. The samples were analyzed via 12% SDS-PAGE under reducing conditions. Lane 1 contains molecular mass standards (approximate values in kDa are presented in lane MW). The other lanes contain, sequentially, CHFI-ER, a control CHFI protein from Z. mays; recombinant CHFI-12345; mutants CHFI-1245, CHFI-2345, CHFI-1234, CHFI-234, and CHFI-123; and EGFP. Each lane contains 2 μg of protein, but 10 μg of EGFP was analyzed. The purification tags of the recombinant proteins were not removed (i.e. N- and/or C-terminal tag with molecular masses of 3.8 and 1.5 kDa, respectively).

FIGURE 3.

Inhibition of trypsin, FXIIa, and FXIa activity by CHFI mutants. A–E, the rate of substrate cleavage by the corresponding protease in reversed coordinates (1/v) on the ordinate axis and depending on the inhibitor concentrations (abscissa axis) obtained in the chromogenic assays. The mean values ± S.E. are plotted. Recombinant EGFP that was prepared under the same experimental conditions as recombinant CHFI was used as a negative control. A, inhibition of FXIIa cleavage of S2302 by native CHFI (CHFI-ER), recombinant CHFI (CHFI-12345), and the mutant variants. B, inhibition of FXIIa cleavage of S2302 cleavage by the synthetic cyclic peptide CHFI-2. The slope is not significant. C, inhibition of FXIa cleavage of S2366 cleavage by native CHFI (CHFI-ER), its mutant CHFI-1245, and the synthetic cyclic peptide CHFI-2. D, inhibition of trypsin cleavage of S2765 by recombinant CHFI (CHFI-12345) and its mutant variants. E, inhibition of trypsin cleavage of S2765 by the synthetic cyclic peptide CHFI-2.

FIGURE 4.

Comparison between the inhibition constants (K_i) of recombinant CHFI (CHFI-12345) and its mutant variants. A–C, K_i values of recombinant (CHFI-12345) CHFI and its mutant variants against FXIIa (A), FXIa (B), and trypsin (C). n/s,5 not significant.

FIGURE 5.

CHFI mutant protein-binding loop conformation, FXIIa model structure, and docked FXIa-CHFI-1245 and FXIIa-CHFI-2 complexes. A and B, conformation of the loop carrying Arg³⁴ in CHFI-1245, as indicated by the molecular dynamics simulation. The salt bridge between Arg³⁴ and Asp²⁹ (A) is shown with dashes. An overlap of the CHFI-12345 (blue) and CHFI-1245 (red) structures after MD simulation is presented in B. C, the angle between α-helixes in CHFI-2 (left) differs from that of CHFI-12345 (right), as indicated by the MD simulation. The axes of α-helixes are shown as cylinders. The structures are visualized using the Bendix plugin (56) for VMD. D, model structure of FXIIa, where catalytic residues are shown as balls and sticks. E and F, protein-protein docking of protease complexes with inhibitor, which is bound tightly to the target far from the active site: FXIa and CHFI-1245 (E) and FXIIa and CHFI-2 (F).

FIGURE 6.

The amino acid sequence of CHFI-2. The loop is rich in prolines (indicated with circles). The asterisk indicates the replacement of Cys²⁹ with Asp. The scissile bond is underlined, and the amino acid positions relative to the reactive site are indicated.