title: Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA).
abstract: 4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers. To understand the high potency and unusual inhibition mechanism of EFdA, we solved RT crystal structures (resolutions from 2.4 to 2.9 Å) that include inhibition intermediates (i) before inhibitor incorporation (catalytic complex, RT/DNA/EFdA-triphosphate), (ii) after incorporation of EFdA-MP followed by dT-MP (RT/DNAEFdA-MP(P)• dT-MP(N) ), or (iii) after incorporation of two EFdA-MPs (RT/DNAEFdA-MP(P)• EFdA-MP(N) ); (iv) the latter was also solved with EFdA-MP mismatched at the N site (RT/DNAEFdA-MP(P)• EFdA-MP(*N) ). We report that the inhibition mechanism and potency of EFdA stem from interactions of its 4'-ethynyl at a previously unexploited conserved hydrophobic pocket in the polymerase active site. The high resolution of the catalytic complex structure revealed a network of ordered water molecules at the polymerase active site that stabilize enzyme interactions with nucleotide and DNA substrates. Finally, decreased translocation results from favorable interactions of primer-terminating EFdA-MP at the pretranslocation site and unfavorable posttranslocation interactions that lead to observed localized primer distortions.
authors.affiliation: C. S. Bond Life Sciences Center, Department of Biochemistry, University of Missouri, Columbia, MO 65211;
authors.forename: Zhe Li
authors.identifier: 0000-0003-2063-1013
authors.initials: ZL
authors.lastname: Salie
authors.affiliation: C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211;
authors.forename: Karen A
authors.initials: KA
authors.lastname: Kirby
authors.affiliation: C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211;
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.affiliation: C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211;
authors.forename: Bruno
authors.initials: B
authors.lastname: Marchand
authors.affiliation: C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211;
authors.forename: Kamalendra
authors.initials: K
authors.lastname: Singh
authors.affiliation: Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261; Magee-Womens Research Institute, University of Pittsburgh, Pittsburgh, PA 15213;
authors.forename: Lisa C
authors.initials: LC
authors.lastname: Rohan
authors.affiliation: Division of Infectious Diseases, Tohoku University and Tohoku Medical Megabank Organization, Sendai 980-8575, Japan;
authors.forename: Eiichi N
authors.initials: EN
authors.lastname: Kodama
authors.affiliation: Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; Department of Infectious Diseases, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan; Department of Hematology, Kumamoto University Graduate School of Medical Sciences, Kumamoto 860-8556, Japan; Research Institute, National Center for Global Health and Medicine, Tokyo 162-8655, Japan; Center for Clinical Sciences, National Center for Global Health and Medicine, Tokyo 162-8655, Japan;
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Parniak
authors.affiliation: C. S. Bond Life Sciences Center, Department of Biochemistry, University of Missouri, Columbia, MO 65211; C. S. Bond Life Sciences Center, Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65211; sarafianoss@missouri.edu.
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003839:Deoxyadenosines / Q000494:pharmacology*; D004795:Enzyme Stability; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
chemical_list: D019380:Anti-HIV Agents; D003839:Deoxyadenosines; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; C558823:islatravir
keywords: EFdA; HIV-1 reverse transcriptase; NRTIs; X-ray crystallography; inhibitors