Interactions of a novel inhibitor from an extremophilic Bacillus sp. with HIV-1 protease: implications for the mechanism of inactivation.

The active site cleft of the HIV-1 protease (PR) is bound by two identical conformationally mobile loops known as flaps, which are important for substrate binding and catalysis. The present article reports, for the first time, an HIV-1 PR inhibitor, ATBI, from an extremophilic Bacillus sp. The inhibitor is found to be a hydrophilic peptide with Mr of 1147, and an amino acid sequence of Ala-Gly-Lys-Lys-Asp-Asp-Asp-Asp-Pro-Pro-Glu. Sequence homology exhibited no similarity with the reported peptidic inhibitors of HIV-1 PR. Investigation of the kinetics of the enzyme-inhibitor interactions revealed that ATBI is a noncompetitive and tight binding inhibitor with the IC(50) and K(i) values 18.0 and 17.8 nm, respectively. The binding of the inhibitor with the enzyme and the subsequent induction of the localized conformational changes in the flap region of the HIV-1 PR were monitored by exploiting the intrinsic fluorescence of the surface exposed Trp-42 residues, which are present at the proximity of the flaps. We have demonstrated by fluorescence and circular dichroism studies that ATBI binds in the active site of the HIV-1 PR and thereby leads to the inactivation of the enzyme. Based on our results, we propose that the inactivation is due to the reorganization of the flaps impairing its flexibility leading toward inaccessibility of the substrate to the active site of the enzyme.