Conformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarnet

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dc.contributor.authorDas, Kalyan
dc.contributor.authorBalzarini, Jan
dc.contributor.authorMiller, Matthew T.
dc.contributor.authorMaguire, Anita R.
dc.contributor.authorDeStefano, Jeffrey J.
dc.contributor.authorArnold, Eddy
dc.contributor.funderScience Foundation Irelanden
dc.contributor.funderNational Institutes of Healthen
dc.contributor.funderKU Leuvenen
dc.date.accessioned2016-11-08T09:38:45Z
dc.date.available2016-11-08T09:38:45Z
dc.date.issued2016-05-18
dc.description.abstractHIV-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.en
dc.description.sponsorshipNational Institutes of Health (Grants R37 AI027690; P50 GM103368; P50GM103368); KU Leuven (GOA 15/19 TBA); Science Foundation Ireland (05/PICA/B802 and SFI 14/TIDA/2402)en
dc.description.statusPeer revieweden
dc.description.versionAccepted Versionen
dc.format.mimetypeapplication/pdfen
dc.identifier.citationDAS, 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.6b00187en
dc.identifier.doi10.1021/acschembio.6b00187
dc.identifier.endpage2164en
dc.identifier.issn1554-8929
dc.identifier.issued8en
dc.identifier.journaltitleACS Chemical Biologyen
dc.identifier.startpage2158en
dc.identifier.urihttps://hdl.handle.net/10468/3253
dc.identifier.volume11en
dc.language.isoenen
dc.publisherAmerican Chemical Societyen
dc.rights© 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.6b00187en
dc.subjectHIV-1 reverse transcriptaseen
dc.subjectNucleotideen
dc.subjectFoscarneten
dc.subjectRTen
dc.titleConformational states of HIV-1 reverse transcriptase for nucleotide incorporation vs. pyrophosphorolysis – binding of foscarneten
dc.typeArticle (peer-reviewed)en
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