title: Mechanism of allosteric inhibition of HIV-1 reverse transcriptase revealed by single-molecule and ensemble fluorescence.
abstract: 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.
authors.affiliation: Program in Molecular Biophysics and Structural Biology, University of Pittsburgh School of Medicine, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213, USA Department of Cell Biology, University of Pittsburgh School of Medicine, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213, USA.
authors.forename: Grant D
authors.initials: GD
authors.lastname: Schauer
authors.affiliation: Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Kelly D
authors.initials: KD
authors.lastname: Huber
authors.affiliation: Program in Molecular Biophysics and Structural Biology, University of Pittsburgh School of Medicine, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213, USA Department of Cell Biology, University of Pittsburgh School of Medicine, Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA 15213, USA leuba@pitt.edu.
authors.forename: Sanford H
authors.initials: SH
authors.lastname: Leuba
authors.affiliation: Department of Medicine, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
mesh_terms: D000480:Alkynes; D000494:Allosteric Regulation; D048588:Benzoxazines / Q000494:pharmacology; D003521:Cyclopropanes; D017931:DNA Primers; D003854:Deoxyribonucleotides / Q000378:metabolism; D005454:Fluorescence Polarization; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D021122:Protein Subunits / Q000737:chemistry; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D013698:Templates, Genetic
chemical_list: D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D017931:DNA Primers; D003854:Deoxyribonucleotides; D021122:Protein Subunits; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; C098320:efavirenz