Non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are routinely used to treat HIV-1 infection, yet their mechanism of action remains unclear despite intensive investigation. In this study, we developed complementary single-molecule fluorescence and ensemble fluorescence anisotropy approaches to discover how NNRTIs modulate the intra-molecular conformational changes and inter-molecular dynamics of RT-template/primer (T/P) and RT-T/P-dNTP complexes. We found that NNRTI binding to RT induces opening of the fingers and thumb subdomains, which increases the dynamic sliding motion of the enzyme on the T/P and reduces dNTP binding affinity. Further, efavirenz promotes formation of the E138-K101 salt bridge between the p51 and p66 subunits of RT, which contributes to opening of the thumb/fingers subdomains. Engineering a more polar salt bridge between p51 and p66 resulted in even greater increases in the thumb/fingers opening, RT sliding, dNTP binding disruption and in vitro and in vivo RT inhibition than were observed with wild-type RT. We also observed that K103N, a clinically relevant NNRTI resistance mutation, does not prevent binding between efavirenz and RT-T/P but instead allows formation of a stable and productive RT-T/P-dNTP complex, possibly through disruption of the E138-K101 salt bridge. Collectively, these data describe unique structure-activity-resistance relationships that could be exploited for drug development.
© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.