title: Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.
abstract: HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.
authors.affiliation: Center for Advanced Biotechnology and Medicine (CABM), Department of Chemistry and Chemical Biology, Rutgers University , Piscataway, New Jersey, United States.
authors.forename: Kalyan
authors.initials: K
authors.lastname: Das
authors.affiliation: Rega Institute for Medical Research and Department of Microbiology and Immunology, KU Leuven , B-3000 Leuven, Belgium.
authors.forename: Jan
authors.initials: J
authors.lastname: Balzarini
authors.affiliation: Center for Advanced Biotechnology and Medicine (CABM), Department of Chemistry and Chemical Biology, Rutgers University , Piscataway, New Jersey, United States.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Miller
authors.affiliation: Department of Chemistry and School of Pharmacy, Analytical and Biological Chemistry Research Facility, Synthesis and Solid State Pharmaceutical Centre, University College Cork , Cork, Ireland.
authors.forename: Anita R
authors.initials: AR
authors.lastname: Maguire
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland College Park , College Park, Maryland, United States.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: DeStefano
authors.affiliation: Center for Advanced Biotechnology and Medicine (CABM), Department of Chemistry and Chemical Biology, Rutgers University , Piscataway, New Jersey, United States.
authors.forename: Eddy
authors.initials: E
authors.lastname: Arnold
mesh_terms: D018360:Crystallography, X-Ray; D011756:Diphosphates / Q000737:chemistry*; D015195:Drug Design; D017245:Foscarnet / Q000737:chemistry*; D054303:HIV Reverse Transcriptase / Q000737:chemistry*; D006868:Hydrolysis; D009711:Nucleotides / Q000737:chemistry*; D011487:Protein Conformation; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry*
chemical_list: D011756:Diphosphates; D009711:Nucleotides; D018894:Reverse Transcriptase Inhibitors; D017245:Foscarnet; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase