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Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.

Conformational States of HIV-1 Reverse Transcriptase for Nucleotide Incorporation vs Pyrophosphorolysis-Binding of Foscarnet.
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Das K, Balzarini J, Miller MT, Maguire AR, DeStefano JJ, Arnold E,


Das K, Balzarini J, Miller MT, Maguire AR, DeStefano JJ, Arnold E, (click to view)

Das K, Balzarini J, Miller MT, Maguire AR, DeStefano JJ, Arnold E,

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ACS chemical biology 2016 06 0611(8) 2158-64 doi 10.1021/acschembio.6b00187

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.

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