Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation

Abstract

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII). Molecular modelling of the CTI-FXIIa complex suggested a canonical inhibitor binding mode. Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction. This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model.

Summary: Background Corn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway. Objectives To investigate the molecular basis of FXII inhibition by CTI. Methods We performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay. Results The docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108-fold, 41-fold, 158-fold, and 100-fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20-44 had inhibitory activity that was three-fold to 4000-fold less than that of full-length CTI. Conclusions The data confirm the validity of a canonical model of the FXIIa-CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Keywords: corn trypsin inhibitor; factor XII; molecular docking simulation; serine protease; trypsin.

Figure 1

Docking of the activated factor XII (FXIIa)–corn trypsin inhibitor (CTI) complex. (A) The canonical inhibitor sequence of CTI loop residues 30–38 is shown aligned to the sequences of the activation loops of FXI, prekallikrein, and FXII which are natural substrates for FXIIa cleavage, and the sequence of peptidomimetic thrombin inhibitor PPACK. (B) Cartoon diagram showing the canonical inhibitor PPACK binding into the S1 pocket of thrombin prepared by use of the crystal structure with Protein Data Bank code 1PPB with pymol. (C) Cartoon diagram of the docked FXIIa–CTI complex. FXIIa is in gray and CTI is in cyan. Key residues are shown as sticks, and interactions are indicated as purple dotted lines. The top boxed area is a zoom‐in of the area of the FXIIa S1, S2 and S3 pockets (gray) and CTI (cyan), and the right‐hand boxed area is the front of the S1 pocket and the FXIIa H1 pocket residue D60 shown interacting with CTI (residue number D60 corresponds to FXII Asp397 and Asp416 in sequence numbering without and with the signal peptide, respectively, or FXII Asp60A from chymotrypsin numbering).

Figure 2

Charge surface representations of the activated factor XII (FXIIa)–corn trypsin inhibitor (CTI) interaction (blue = positive, red = negative). (A) The FXIIa model is shown with the key surface pockets labeled. (B) Docked complex of FXIIa (shown as a cartoon in gray) and CTI shown as a transparent charged surface illustrating the basic region interacting with the negatively charged S1 (D189) and H1 (D60) residues shown as sticks. (C) FXIIa protease as viewed in (A) but with CTI residues 20–44 shown as a cartoon in cyan, with key residues labeled as sticks in cyan.

Figure 3

Expression of recombinant corn trypsin inhibitor (CTI) mutants and activated factor XII (α‐FXIIa) activity in the presence of recombinant CTI (rec‐CTI), commercial CTI, and glutathione‐S‐transferase (GST). (A) Wild‐type CTI or mutants were expressed as His‐GST fusion proteins, as explained in Materials and methods. Purified proteins were quantified by the Lowry method, and 1 μg of protein was analyzed by SDS‐PAGE followed by Coomassie blue staining. (B) CTI samples (10⁻⁸ m to 10⁻⁵ m) were incubated with 200 μm substrate peptide, and this was followed by addition of α‐FXIIa; enzymatic activity was then monitored as described in Materials and methods. Data points were fitted to a curve by non‐linear regression (graphpad prism 6.04; log[inhibitor] versus response – variable slope algorithm with a bottom constraint). Error bars indicate the standard error (n = 3–5 independent observations).

Figure 4

Inhibition of activated factor XII (α‐FXIIa) by recombinant corn trypsin inhibitor (rec‐CTI) variants and peptides. Concentrations of rec‐CTI variants (10⁻⁸ m to 10⁻⁵ m) or peptides (10⁻⁶ m to 10⁻³ m) were incubated with 200 μm substrate peptide, and this was followed by addition of α‐FXIIa; enzymatic activity was then monitored as described in Materials and methods. pIC ₅₀ values were obtained by non‐linear regression (graphpad prism 6.04; log[inhibitor] versus response – variable slope algorithm with a bottom constraint). Error bars indicate the standard error (n = 3–5 independent observations).

Figure 5

Corn trypsin inhibitor (CTI) substitutions affect inhibition of activated factor XII (FXIIa) to different degrees. (A) The CTI amino acid sequence for residues 20–44 with the respective contact pockets in FXIIa indicated above. (B) Cartoon diagram of the CTI structure showing residues affected by the mutagenesis experiments. (C) Close‐up view of the structure of CTI residues 20–44. Color coding in B and C: Red ‐ essential for binding; Green ‐ important for binding; Orange ‐ contributes to binding.

Figure 6

A model for corn trypsin inhibitor (CTI) protease selectivity. (A) Superposition of the activated factor XII (FXIIa) model (purple) and trypsin (gray) shown as a Cα backbone in the region of the S1 and S3 pockets, illustrating the main chain similarity of the 99‐loop. Key side chains are shown as sticks for trypsin (orange) and FXIIa (purple). (B) A combination of features around the S1 and S3 pockets are more open for the bulky CTI inhibitor to bind, with shape changes in the S1 and S3 pockets of other proteases preventing binding of CTI by steric occlusion.