Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence

Nucleic Acids Res. 2014 Oct;42(18):11687-96. doi: 10.1093/nar/gku819. Epub 2014 Sep 17.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkynes
  • Allosteric Regulation
  • Benzoxazines / pharmacology
  • Cyclopropanes
  • DNA Primers
  • Deoxyribonucleotides / metabolism
  • Fluorescence Polarization
  • HIV Reverse Transcriptase / antagonists & inhibitors*
  • HIV Reverse Transcriptase / chemistry*
  • HIV Reverse Transcriptase / genetics
  • HIV Reverse Transcriptase / metabolism
  • Mutation
  • Protein Subunits / chemistry
  • Reverse Transcriptase Inhibitors / pharmacology*
  • Templates, Genetic


  • Alkynes
  • Benzoxazines
  • Cyclopropanes
  • DNA Primers
  • Deoxyribonucleotides
  • Protein Subunits
  • Reverse Transcriptase Inhibitors
  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase
  • efavirenz