Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarnet
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Accepted Version
Date
2016-05-18
Authors
Das, Kalyan
Balzarini, Jan
Miller, Matthew T.
Maguire, Anita R.
DeStefano, Jeffrey J.
Arnold, Eddy
Journal Title
Journal ISSN
Volume Title
Publisher
American Chemical Society
Published Version
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 Mg2+ 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 Mg2+ (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.
Description
Keywords
HIV-1 reverse transcriptase , Nucleotide , Foscarnet , RT
Citation
DAS, K., BALZARINI, J., MILLER, M. T., MAGUIRE, A. R., DESTEFANO, J. J. and ARNOLD, E. (2016) ‘Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarnet’, ACS Chemical Biology, 11(8), pp. 2158-2164. doi:10.1021/acschembio.6b00187
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Copyright
© 2016, American Chemical Society. This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Chemical Biology, copyright © American Chemical Society, after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/acschembio.6b00187