Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

Chavali, Sai Shashank; Bonn-Breach, Rachel; Wedekind, Joseph E.

doi:10.1074/jbc.rev119.006860

Cited by 38 publications

(44 citation statements)

References 157 publications

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“…These inhibitors recognize HIV TAR with KD values in the nanomolar to picomolar range, while yielding Ki values of ~40 μM in antiviral assays (23). Importantly, NMR analysis of an ultrahighaffinity inhibitor from this family revealed TAR recognition at two guanine nucleobases by specific arginines (23), demonstrating parallels to the natural modes of TAR and 7SK recognition by the Tat ARM (15). These observations pose the question of whether peptide inhibitors could be developed that use three or more arginines to target the Hoogsteen edges of conserved guanines in HIV TAR, thereby imparting even greater specificity.…”

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confidence: 95%

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

Chavali

Mali

Jenkins

et al. 2020

Journal of Biological Chemistry

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RNA-protein interfaces control key replication events during the HIV-1 lifecycle. The viral trans-activator of transcription (Tat) protein uses an archetypal ARM (arginine-rich motif) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 nM and 3.0 ± 0.3 nM). Our efforts yielded low molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6-22 μM). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

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confidence: 95%

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

Chavali

Mali

Jenkins

et al. 2020

Journal of Biological Chemistry

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“…Identifying and understanding these networks remains a central challenge in the ncRNA field ( 1–4 ). Riboswitches are ideal systems to explore ligand-induced ncRNA conformational changes due to their highly selective recognition of cognate effectors ( 5 ), a typically two-domain organization that features a conserved aptamer connected to a gene-regulatory expression platform ( 6 , 7 ), and their tractability for high-resolution structural analysis ( 8 , 9 ). Representative co-crystal structures have been determined for >30 distinct riboswitch classes bound to their cognate effectors ( 9 ), as well as to natural and synthetic compounds ( 10–14 ).…”

Section: Introductionmentioning

confidence: 99%

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation

Schroeder

Dutta

Cavender

et al. 2020

Nucleic Acids Research

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Riboswitches are structured RNA motifs that recognize metabolites to alter the conformations of downstream sequences, leading to gene regulation. To investigate this molecular framework, we determined crystal structures of a preQ1-I riboswitch in effector-free and bound states at 2.00 Å and 2.65 Å-resolution. Both pseudoknots exhibited the elusive L2 loop, which displayed distinct conformations. Conversely, the Shine-Dalgarno sequence (SDS) in the S2 helix of each structure remained unbroken. The expectation that the effector-free state should expose the SDS prompted us to conduct solution experiments to delineate environmental changes to specific nucleobases in response to preQ1. We then used nudged elastic band computational methods to derive conformational-change pathways linking the crystallographically-determined effector-free and bound-state structures. Pathways featured: (i) unstacking and unpairing of L2 and S2 nucleobases without preQ1—exposing the SDS for translation and (ii) stacking and pairing L2 and S2 nucleobases with preQ1—sequestering the SDS. Our results reveal how preQ1 binding reorganizes L2 into a nucleobase-stacking spine that sequesters the SDS, linking effector recognition to biological function. The generality of stacking spines as conduits for effector-dependent, interdomain communication is discussed in light of their existence in adenine riboswitches, as well as the turnip yellow mosaic virus ribosome sensor.

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“…The TAR element is a 59-nt sequence located at the 5′-end of all HIV-1 nascent viral transcripts and plays an essential role in Tat-mediated transcriptional activation [ 244 , 245 , 246 , 247 , 248 ]. Mutagenesis studies led to proposals that TAR is also involved in a variety of functions, including dimerization [ 249 , 250 ], strand transfer during reverse transcription [ 251 ], translation [ 75 ], packaging [ 239 , 252 , 253 , 254 ], HIV-1 derived microRNA (miRNA) during latency [ 255 ], and even the growth and progression of some cancers [ 256 ].…”

Section: Rna Components Important For Genome Packagingmentioning

confidence: 99%

NMR Studies of Retroviral Genome Packaging

Boyd

Brown

et al. 2020

Viruses

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Nearly all retroviruses selectively package two copies of their unspliced RNA genomes from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Over the past four decades, combinations of genetic experiments, phylogenetic analyses, nucleotide accessibility mapping, in silico RNA structure predictions, and biophysical experiments were employed to understand how retroviral genomes are selected for packaging. Genetic studies provided early clues regarding the protein and RNA elements required for packaging, and nucleotide accessibility mapping experiments provided insights into the secondary structures of functionally important elements in the genome. Three-dimensional structural determinants of packaging were primarily derived by nuclear magnetic resonance (NMR) spectroscopy. A key advantage of NMR, relative to other methods for determining biomolecular structure (such as X-ray crystallography), is that it is well suited for studies of conformationally dynamic and heterogeneous systems—a hallmark of the retrovirus packaging machinery. Here, we review advances in understanding of the structures, dynamics, and interactions of the proteins and RNA elements involved in retroviral genome selection and packaging that are facilitated by NMR.

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Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

Cited by 38 publications

References 157 publications

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

Analysis of a preQ1-I riboswitch in effector-free and bound states reveals a metabolite-programmed nucleobase-stacking spine that controls gene regulation

NMR Studies of Retroviral Genome Packaging

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