Interactions Outside the Proteinase-Binding Loop Contribute Significantly to the Inhibition of Activated Coagulation Factor XII by its Canonical Inhibitor from Corn [Enzymology]

April 4th, 2014 by Korneeva, V. A., Trubetskov, M. M., Korshunova, A. V., Lushchekina, S. V., Kolyadko, V. N., Sergienko, O. V., Lunin, V. G., Panteleev, M. A., Ataullakhanov, F. I.

Activated factor XII (FXIIa) is selectively inhibited by corn Hageman factor inhibitor (CHFI) and 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 (1BEA in the Protein Data Bank (PDB)), 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 (FXIa) (Ki=94 ± 11 μM). Full-length CHFI inhibited trypsin with a Ki of 1.3 ± 0.2 μM and FXIa 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.
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