Michael F. Bardaro

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

The HIV-1 TAR RNA represents a well-known paradigm to study the role of dynamics and conformational change in RNA function. This regulatory RNA changes conformation in response to binding of Tat protein and of a variety of peptidic and small molecule ligands, indicating that its conformational flexibility and intrinsic dynamics play important roles in molecular recognition. We have used 13C NMR relaxation experiments to examine changes in the motional landscape of HIV-1 TAR in the presence of three ligands of different affinity and specificity. The ligands are argininamide, a linear peptide mimic of the Tat basic domain and a cyclic peptide that potently inhibits Tat-dependent activation of transcription. All three molecules induce the same motional characteristics within the three nucleotides bulge that represents the Tat-binding site. However, the cyclic peptide has a unique motional signature in the apical loop, which represents a binding site for the essential host co-factor cyclin T1. These results suggest that all peptidic mimics of Tat induce the same dynamics in TAR within this protein binding site. However, the new cyclic peptide mimic of Tat represents a new class of ligands with a unique effect on the dynamics and the structure of the apical loop.

Intermediate Rate Atomic Trajectories of RNA by Solid-State NMR Spectroscopy

Many RNAs undergo large conformational changes in response to the binding of proteins and small molecules. However, when RNA functional dynamics occur in the nanosecond-microsecond time scale, they become invisible to traditional solution NMR relaxation methods. Residual dipolar coupling methods have revealed the presence of extensive nanosecond-microsecond domain motions in HIV-1 TAR RNA, but this technique lacks information on the rates of motions. We have used solid-state deuterium NMR to quantitatively describe trajectories of key residues in TAR by exploiting the sensitivity of this technique to motions that occur in the nanosecond-microsecond regime. Deuterium line shape and relaxation data were used to model motions of residues within the TAR binding interface. The resulting motional models indicate two functionally essential bases within the single-stranded bulge sample both the free and Tat-bound conformations on the microsecond time scale in the complete absence of the protein. Thus, our results strongly support a conformational capture mechanism for recognition: the protein does not induce a new RNA structure, but instead captures an already-populated conformation.

Solid-State Deuterium NMR Studies Reveal μs-ns Motions in the HIV-1 Transactivation Response RNA Recognition Site

Solution and solid-state NMR measurements were used together to examine motion in three sites in the HIV-1 TAR RNA. We wished to investigate the dynamics facilitating the conformational rearrangements the TAR RNA must undergo for Tat binding and in particular to characterize the full range of motional time scales accessible to this RNA. Our results demonstrate that the dynamics in TAR involving residues essential to Tat binding include not only the faster motions detected by solution relaxation measurements but also a significant component in the μs-ns time scale.

Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in 2H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state 13C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.

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

Significance All biochemical reactions in living organisms require molecular recognition events. In particular, the interactions between protein and RNA molecules are crucial in the regulation of gene expression. However, the transient nature of the conformations populated during the recognition process has prevented a detailed characterization of the mechanisms by which these interactions take place. To address this problem, we report a high-resolution structure of an intermediate state in protein-RNA recognition. We determined this structure by using NMR measurements as ensemble-averaged structural restraints in metadynamics simulations, and validated it by performing a structure-based design of two mutants with rationally modified binding rates. 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.

A novel small-molecule binds to the influenza A virus RNA promoter and inhibits viral replication

Through screening by NMR spectroscopy, we discovered a novel scaffold (DPQ: 6,7-dimethoxy-2-(1-piperazinyl)-4-quinazolinamine) that binds specifically to the influenza A virus RNA promoter. The solution structure of the RNA-DPQ complex reported here demonstrates that the internal loop is the binding site of DPQ. The scaffold exhibits antiviral activity against influenza viruses.

Examining the relationship between RNA function and motion using nuclear magnetic resonance

The biological function of proteins and nucleic acids relies on their complex structures, yet dynamics provides an additional layer of functional adaptability. Numerous studies have demonstrated that RNA is only able to perform the multitude of functions for which it is responsible by readily changing its conformation in response to binding of proteins or small molecules. Examination of RNA dynamics is therefore essential to understanding its biological function. Nuclear magnetic resonance (NMR) has emerged as a leading technique for the examination of RNA motion and conformational transitions. It can examine domain motions as well as motion with atomic level resolution over a wide range of time scales. This review examines how NMR spectroscopy can be applied to examine the relationship between function and dynamics in RNA. WIREs RNA 2012, 3:122–132. doi: 10.1002/wrna.108

Independent alignment of RNA for dynamic studies using residual dipolar couplings

Molecular motion and dynamics play an essential role in the biological function of many RNAs. An important source of information on biomolecular motion can be found in residual dipolar couplings which contain dynamics information over the entire ms-ps timescale. However, these methods are not fully applicable to RNA because nucleic acid molecules tend to align in a highly collinear manner in different alignment media. As a consequence, information on dynamics that can be obtained with this method is limited. In order to overcome this limitation, we have generated a chimeric RNA containing both the wild type TAR RNA, the target of our investigation of dynamics, as well as the binding site for U1A protein. When U1A protein was bound to the portion of the chimeric RNA containing its binding site, we obtained independent alignment of TAR by exploiting the physical chemical characteristics of this protein. This technique can allow the extraction of new information on RNA dynamics, which is particularly important for time scales not covered by relaxation methods where important RNA motions occur.

Elucidating molecular motion through structural and dynamic filters of energy-minimized conformer ensembles.

Complex RNA structures are constructed from helical segments connected by flexible loops that move spontaneously and in response to binding of small molecule ligands and proteins. Understanding the conformational variability of RNA requires the characterization of the coupled time evolution of interconnected flexible domains. To elucidate the collective molecular motions and explore the conformational landscape of the HIV-1 TAR RNA, we describe a new methodology that utilizes energy-minimized structures generated by the program “Fragment Assembly of RNA with Full-Atom Refinement (FARFAR)”. We apply structural filters in the form of experimental residual dipolar couplings (RDCs) to select a subset of discrete energy-minimized conformers and carry out principal component analyses (PCA) to corroborate the choice of the filtered subset. We use this subset of structures to calculate solution T1 and T1ρ relaxation times for 13C spins in multiple residues in different domains of the molecule using two simulation protocols that we previously published. We match the experimental T1 times to within 2% and the T1ρ times to within less than 10% for helical residues. These results introduce a protocol to construct viable dynamic trajectories for RNA molecules that accord well with experimental NMR data and support the notion that the motions of the helical portions of this small RNA can be described by a relatively small number of discrete conformations exchanging over time scales longer than 1 μs.

Correction: A novel small-molecule binds to the influenza A virus RNA promoter and inhibits viral replication

Correction for ‘A novel small-molecule binds to the influenza A virus RNA promoter and inhibits viral replication’ by Mi-Kyung Lee et al., Chem. Commun., 2014, 50, 368–370.