Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function of inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed curled/tucked, closed, semi-open and wide-open conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (Pis) as well as a non hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.