Macromolecular inhibitors of HIV-1 protease - Characterization of designedheterodimers

Defective variants of human immunodeficiency virus type 1 (HIV-1) protease (HIV PR) have been engineered to inhibit wild-type (wt) PW PR activity. The se variants were designed to promote the formation of heterodimers and to d estabilize the formation of inactive variant homodimers of HIV-1 protease t hrough substitutions at Asp-25, Ile-49, and Gly-50 (Babe, L, M., Rose, J., and Craik, C, S. (1995) Proc. Natl, Acad Sci. U.S.A. 92, 10069-10073; McPhe e, F., Good, A. C., Kuntz, I. D., and Craik, C. S. (1996) Proc. Natl. Acad Sci. U.S.A. 93, 11477-11481). The mechanism of action of these dominant-neg ative inhibitors was established using recombinantly expressed defective mo nomers, The defective monomers were refolded in vitro in the presence of wt HIV PR and showed dose-dependent inhibition of proteolytic activity. This inhibition was shown to result from the formation of inactive heterodimers between defective and wt HIV PR monomers, Heterodimer formation was detecte d by (i) isolating refolded, inactive heterodimers using histidine-tagged d efective monomers and (ii) isolating heterodimers from bacteria coexpressin g both wt and defective variants of HIV PR. Single-chain variants of HIV PR , in which the C terminus of the wt HIV PR monomer was covalently tethered to the N terminus of the defective monomer, were also expressed and analyze d. Thermal denaturation of these single-chain heterodimers using differenti al scanning calorimetry revealed a 1.5-7.2 degrees C greater thermal stabil ity than single-chain wt HIV PR, The thermodynamic trend shown by these thr ee variants mirrors their relative inhibition in provirus transfection assa ys. These data support the model that the effects seen both in tissue cultu re and in vitro arise from an increase in stability conferred on these hete rodimers by interface mutations and identifies heterodimer formation as the ir mechanism of inhibition.