Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA

Sarafianos, Stefan G.; Das, Kalyan; Tantillo, Chris; Clark, Arnold M.; Ding, Jianping; Whitcomb, Jeannette M.; Boyer, Paul L.; Hughes, Stephen H.; Arnold, Edward

doi:10.1093/emboj/20.6.1449

Cited by 387 publications

(673 citation statements)

References 77 publications

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“…3b), suggesting that the interactions between RT and nucleic acid at opposite ends of the primer-template binding cleft were most important in determining the binding orientation. This observation is consistent with the crystal structures, which show RT-substrate contacts primarily clustered in two regions near the DNA polymerase and RNase H active sites 19,22 . A single nucleotide provided the strongest determinant of binding orientation: changing the sugar content of the fifth nucleotide from the primer 59 terminus alone caused a nearly 2k B T (where k B is the Boltzmann constant) change in DG (Fig.…”

supporting

confidence: 91%

“…3). Taken together, these results indicate that RT binds to the DNA-DNA primer-template complex with its polymerase active site between the fingers and palm domains close to the 39 end of the primer and the RNase H domain near the 59 end-an orientation that matches the polymerization-competent binding mode observed in RT-substrate co-crystal structures [19][20][21][22] . A virtually identical binding orientation was observed for RT on a 19-nt DNA primer annealed to a 50-nt RNA template ( Supplementary Fig.…”

supporting

confidence: 62%

“…The mechanism by which RT discriminates between these substrates and executes the appropriate catalytic function is, however, poorly understood. Although RNase H cleavage analysis suggests the presence of different interaction modes of RT with substrates 16,17 , crystal structures have so far revealed only one enzyme-binding orientation 4,[18][19][20][21][22] .…”

mentioning

confidence: 99%

“…Static FRET measurements have also been used previously to characterize the pre-translocation and post-translocation states of RT on a DNA duplex 28 . Because RT accommodates 19-22 base pairs of nucleic-acid duplex within its primer-template-binding cleft 19,22,29 (Fig. 1a), we constructed several duplex substrates with different backbone compositions and base sequences, each consisting of a 50-nucleotide (nt) oligonucleotide mimicking the template and a complementary 19-21-nt oligonucleotide emulating the primer (Fig.…”

mentioning

confidence: 99%

“…This position makes specific contacts with RT residues T473 and Q475 located within the RNase H primer grip 19,22 . These residues are conserved between RNases H found in viruses, bacteria and humans 22,31,32 . Replacement of these residues with alanine in HIV-1 RT decreases the DNA synthesis rate and inhibits virus infectivity 33 .…”

mentioning

confidence: 99%

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Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

173

216

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The reverse transcriptase of human immunodeficiency virus (HIV) catalyses a series of reactions to convert the single-stranded RNA genome of HIV into double-stranded DNA for host-cell integration. This task requires the reverse transcriptase to discriminate a variety of nucleic-acid substrates such that active sites of the enzyme are correctly positioned to support one of three catalytic functions: RNA-directed DNA synthesis, DNA-directed DNA synthesis and DNA-directed RNA hydrolysis. However, the mechanism by which substrates regulate reverse transcriptase activities remains unclear. Here we report distinct orientational dynamics of reverse transcriptase observed on different substrates with a single-molecule assay. The enzyme adopted opposite binding orientations on duplexes containing DNA or RNA primers, directing its DNA synthesis or RNA hydrolysis activity, respectively. On duplexes containing the unique polypurine RNA primers for plus-strand DNA synthesis, the enzyme can rapidly switch between the two orientations. The switching kinetics were regulated by cognate nucleotides and non-nucleoside reverse transcriptase inhibitors, a major class of anti-HIV drugs. These results indicate that the activities of reverse transcriptase are determined by its binding orientation on substrates.Virtually all RNA-processing and DNA-processing enzymes show selectivity for backbone compositions or base sequences of their nucleic-acid substrates. This substrate selectivity is especially crucial for the HIV-1 reverse transcriptase (RT), which binds and discriminates between a variety of nucleic-acid duplexes for distinct catalytic functions 1,2 . RT is a heterodimer consisting of a p51 and a p66 subunit, the latter of which contains catalytically active DNA polymerase and RNase H domains 3,4 , catalysing a complex, multi-step reaction to convert the single-stranded RNA genome into double-stranded DNA 1,2 . First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA 11-13 , thereby creating the double-stranded DNA viral genome. Specific cleavage by RNase H then removes the PPT primers and exposes the integration sequence to facilitate the insertion of the viral DNA into the host chromosome 14 . Inappropriate initiation of synthesis of the plus-strand DNA at other RNA segments prevents integration 2,15 . RT must therefore obey the following primer-selection rules: first, DNA primers readily engage the polymerase activity of RT; second, generic RNA primers are not efficiently extended by RT but readily engage the RNase H activity of RT when annealed with DNA; third, the PPT RNA ca...

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supporting

confidence: 91%

supporting

confidence: 62%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Dynamic binding orientations direct activity of HIV reverse transcriptase

Abbondanzieri¹,

Bokinsky²,

Rausch

et al. 2008

Nature

173

216

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X‐Ray Crystallography in Drug Discovery

Livingston¹,

Buchanan²,

D’Amico³

et al. 2003

Burger's Medicinal Chemistry and Drug Discovery

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The focus of this chapter is the methods, recent advances, and the applications of X‐ray protein crystallography in the discovery of new drug targets and candidate drugs. It begins with a basic description of the various steps involved in solving a structure, including recent advances in technique and technology, and the major challenges that are faced in executing the process. The underlying theory, various crystallization methods, data collection equipment and techniques, and computational reduction of the data to give the final structure are all covered in this section. The accessible databases that contain large collections of structures are also described. The application to drug discovery is then briefly addressed with a discussion of co‐crystallization methods and issues, which is followed by a comprehensive table enumerating the published structures of the known drug targets and close homologs. Applications to discovering and validating protein targets of therapeutic relevance are then covered in a section on the very new field of structural genomics. Genome annotation by structural homology, elucidating pathways and the potential for the rational design of multitarget drugs, and advances in the field of protein structure modeling that are enabled by crystallographic databases are described and exemplified with very recent results from the field.

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HIVReverse Transcriptase Inhibitors

Clercq

2010

Burger's Medicinal Chemistry and Drug Discovery

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The first antiviral compound ever shown to be active in vitro against HIV (human immunodeficiency virus) (originally referred to as HTLV‐III (human lymphotropic virus type III) and LAV‐1 (lymphadenopathy‐associated virus type 1)) was suramin; suramin was also the first antiviral found to be effective in vivo in the treatment of HIV infections. Suramin was explored as a potential strategy for the therapy of AIDS because it has been recognized as a potent inhibitor of the reverse transcriptase (RT) associated with a number of animal retroviruses. Later, it became evident that suramin primarily acts against HIV infections as an inhibitor of virus adsorption rather than an RT inhibitor.

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Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA

Cited by 387 publications

References 77 publications

Dynamic binding orientations direct activity of HIV reverse transcriptase

Dynamic binding orientations direct activity of HIV reverse transcriptase

X‐Ray Crystallography in Drug Discovery

HIVReverse Transcriptase Inhibitors

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