How binding of small molecule and peptide ligands to HIV-1 TAR alters the RNA motional landscape

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165

226

The interaction of the HIV-1 transactivator protein Tat with its transactivation response (TAR) RNA is an essential step in viral replication and therefore an attractive target for developing antivirals with new mechanisms of action. Numerous compounds that bind to the 3-nt bulge responsible for binding Tat have been identified in the past, but none of these molecules had sufficient potency to warrant pharmaceutical development. We have discovered conformationally-constrained cyclic peptide mimetics of Tat that are specific nM inhibitors of the Tat-TAR interaction by using a structure-based approach. The lead peptides are nearly as active as the antiviral drug nevirapine against a variety of clinical isolates in human lymphocytes. The NMR structure of a peptide-RNA complex reveals that these molecules interfere with the recruitment to TAR of both Tat and the essential cellular cofactor transcription elongation factor-b (P-TEFb) by binding simultaneously at the RNA bulge and apical loop, forming an unusually deep pocket. This structure illustrates additional principles in RNA recognition: RNA-binding molecules can achieve specificity by interacting simultaneously with multiple secondary structure elements and by inducing the formation of deep binding pockets in their targets. It also provides insight into the P-TEFb binding site and a rational basis for optimizing the promising antiviral activity observed for these cyclic peptides.NMR ͉ transcription elongation factor-b ͉ antiviral ͉ Tat-TAR interaction ͉ RNA recognition T ranscription of the HIV-1 RNA in infected cells is strongly activated by the complex between the viral Tat protein and its cognate transactivation response (TAR) RNA, a 59-nt RNA found at the 5Ј end of all nascent viral transcripts (Fig. 1A). Tat and its cellular cofactor, the transcription elongation factor-b (P-TEFb), are recruited to the elongating RNA polymerase II (RNAP II) through interactions with TAR and are required for reactivation of the integrated proviral genome in latently infected cells (1). The cooperative binding of Tat and P-TEFb to TAR activates the CDK9 kinase of P-TEFb that phosphorylates RNAP II and the repressive NELF factors, leading to greatly enhanced RNAP II processivity (1-3).The Tat-TAR complex is an attractive target for developing new antivirals because the interaction between Tat and TAR is unique to the virus, whereas P-TEFb is used widely for transcription of most host genes. Furthermore, TAR is extremely conserved among viral isolates and P-TEFb plays a key role in promoting infectivity through the Tat-TAR complex and in the emergence from latency. These considerations have led to the synthesis and evaluation of numerous small-molecule and peptidic inhibitors of the Tat-TAR interaction during the last 15 years (4-7). However, none of these molecules had sufficient potency or selectivity to progress into preclinical studies and, indeed, inhibiting the Tat-TAR interaction has proven challenging. First, there is little precedent for the pharmacological disruption...

Section: Structure Of the Tar-l-22supporting

confidence: 60%

Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein

Davidson

Leeper

Athanassiou

et al. 2009

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165

226

“…6). This could be due to secondary binding to the apical loop (45,46) especially given the high concentration of ARG used in our study. Thus, although we observe the expected quenching of relaxation dispersion at slow-exchanging sites in the apical loop, we cannot rule out that this arises due to direct binding of ARG to the apical loop.…”

Section: G34-c8mentioning

confidence: 92%

Invisible RNA state dynamically couples distant motifs

Lee

Dethoff

Al‐Hashimi

2014

113

Using on-and off-resonance carbon and nitrogen R1ρ NMR relaxation dispersion in concert with mutagenesis and NMR chemical shift fingerprinting, we show that the transactivation response element RNA from the HIV-1 exists in dynamic equilibrium with a transient state that has a lifetime of ∼2 ms and population of ∼0.4%, which simultaneously remodels the structure of a bulge, stem, and apical loop. This is accomplished by a global change in strand register, in which bulge residues pair up with residues in the upper stem, causing a reshuffling of base pairs that propagates to the tip of apical loop, resulting in the creation of three noncanonical base pairs. Our results show that transient states can remodel distant RNA motifs and possibly give rise to mechanisms for rapid long-range communication in RNA that can be harnessed in processes such as cooperative folding and ribonucleoprotein assembly.NMR spectroscopy | dynamics | R1ρ relaxation dispersion | nucleic acids I t is now well-established that RNA sequences do not code for a single static structure, but rather, many conformations that populate energetic minima along a free-energy landscape (1, 2). Cellular inputs, ranging from changes in temperature and pH to the binding of proteins, other RNAs, and ligands, can preferentially stabilize select conformations along the landscape, resulting in dynamic changes in RNA structure that drive the multistep catalytic cycles of ribozymes (3), regulatory activities of riboswitches (4) and other RNA-based switches (5), and the dynamic assembly and disassembly of ribonucleoprotein (RNP) complexes (6).A common mode of RNA dynamics involves rearrangements in secondary structure that can melt or create entire hairpins, and thereby expose or sequester key regulatory elements that are several nucleotides long (1,4,7,8). Such secondary structural transitions entail large kinetic barriers, so they are often catalyzed by RNA-binding proteins (9), ATP-dependent chaperones (10), or otherwise occur by modulating cotranscriptional folding (5, 11). Recently, NMR R1ρ relaxation dispersion experiments (12-15) in concert with mutagenesis (16) have helped uncover more labile RNA secondary structural transitions that can take place without assistance from external cofactors at rates that are 2-4 orders of magnitude faster than larger-scale secondary structural rearrangements. These transitions entail excursions away from the energetically favorable ground state (GS) toward lowpopulated (typically populations <15%) and short-lived (lifetime < milliseconds) species often referred to as "excited states" (ES) (12, 13). These invisible RNA ES feature localized reshuffling of base pairing in and around noncanonical motifs such as bulges, internal loops, and apical loops (16) which can also expose or sequester functionally important residues or promote ATPindependent large-scale changes in secondary structure (14, 15). These faster and more localized changes in secondary structure may meet unique demands in RNA-based regulatory functions (16).Using...

“…The HIV TAR-Tat interaction has been a subject of major attention in the past two decades, both for understanding the mechanism of transactivation and for development of anti-HIV therapeutics (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)30), and as a paradigm for the mechanism underlying protein-RNA recognition and signaling observed in a wide range of posttranscriptional regulatory processes. Our results reveal the structure of an intermediate in this interaction, illustrating how the use of RDCs as structural restraints in RAM simulations, particularly with further experimental validation through structure-based mutant design, provides a general strategy for obtaining high-resolution structures of low-population intermediates of RNA-protein complexes, which are very challenging for more conventional structure determination or dynamic techniques.…”

Section: Discussionmentioning

confidence: 99%

“…1 and Fig. S1) with a highly dynamic structure (10,12,13). The NMR structures of free TAR (14)(15)(16) and of TAR bound to peptide fragments of Tat and to peptide mimetics of Tat in HIV (16)(17)(18)(19)(20) and other lentiviruses (21,22) revealed the conformational properties of TAR in its free and bound states, and demonstrated that this RNA molecule undergoes significant dynamic rearrangements associated with its functions.…”

mentioning

confidence: 91%

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Structure of a low-population binding intermediate in protein-RNA recognition

Borkar

Bardaro

Camilloni

et al. 2016

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The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution.RNA structure | NMR spectroscopy | metadynamics | exact RDC restraints | tensor-free method E ssentially all biochemical reactions taking place in living organisms are associated with macromolecular recognition events. A full understanding the molecular mechanisms underlying such events requires the characterization of binding intermediates, which are states that typically have lifetimes of less than a millisecond and may comprise only 5-15% of the conformational space of proteins (1) and nucleic acids (2, 3). Protein-protein and protein-DNA intermediates have recently been characterized at high resolution (4, 5), but despite considerable advances (3, 6-8), high-resolution structures for protein-RNA intermediates have not been reported yet.To address this problem, we focused on the well-studied process by which HIV, like other lentiviruses, hijacks the host transcription machinery to activate transcription of the viral genome (9-13). In HIV, transactivation (Fig. S1) requires binding of the transactivator of transcription (Tat) protein and the host positive transcription elongation factor b (P-TEFb) complex (11) to the transactivation response element (TAR), a 59-residue RNA stem-loop ( Fig. 1 and Fig. S1) with a highly dynamic structure (10, 12, 13). The NMR structures of free TAR (14-16) and of TAR bound to peptide fragments of Tat and to peptide mimetics of and other lentiviruses (21,22) revealed the conformational properties of TAR in its free and bound states, and demonstrated that this RNA molecule undergoes significant dynamic rearrangements associated with its functions. Although the TAR-Tat complex has become a paradigm for the widespread occurrence of conformational rearrangements and molecular adaptation in protein-RNA recognition, the pathway and intermediates linking the free and bound states of TAR are still unknown. Results and DiscussionDetermination of the Tat-TAR Free Energy Landscape. Following an approach recently described for proteins (4) we ident...