Double Electron-Electron Resonance and 1H-15N HSQC Spectral Analysis as Means to Understand the Effects of Single and Multiple Site Mutations in the HIV-1 protease

Abstract

In this study, site-directed spin labeling coupled with electron paramagnetic resonance (SDSL-EPR) was used to probe changes in the conformational sampling of subtype B human immunodeficiency virus type 1 protease (HIV-1PR) attributed to the active site mutation D30N and the non-active site mutations M36I and A71V. Double electron-electron resonance (DEER) was employed to derive distance distribution profiles for each MTSL-labeled construct. Those with double mutations (D30N M36I and D30N A71V) exhibited higher percentage of the closed conformation while that with triple mutation (D30N M36I A71V) has a higher percentage of wide-open conformation relative to wild-type. The percentage semi-open conformation markedly decreased in the former constructs while it is comparable to wild-type for the latter.Meanwhile, 1H-15N heteronuclear single quantum coherence (HSQC) spectra were obtained for HIV-1PR constructs with single (M36I), double (D30N M36I) and triple mutations (D30N M36I A71V), and were compared to that of the wild-type. HIV-1PR residues that have marked chemical shift perturbations as a result of an active or non-active site mutation were identified. Non-active site mutations occurring at the flap elbow (M36I) or beta-sheet core (A71V) of the protease were found to affect chemical shift more than the mutation at the active site pocket (D30N). Information gained from single-point mutation-induced changes in the conformational sampling and dynamics of the HIV-1PR may provide key insights on the mechanism of drug resistance.