RESUMO
Understanding small molecule binding to RNA can be complicated by an intricate interplay between binding stoichiometry, multiple binding motifs, different occupancies of different binding motifs, and changes in the structure of the RNA under study. Here, we use native top-down mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy to experimentally resolve these factors and gain a better understanding of the interactions between neomycin B and the 40 nt aptamer domain of a neomycin-sensing riboswitch engineered in yeast. Data from collisionally activated dissociation of the 1:1, 1:2 and 1:3 RNA-neomycin B complexes identified a third binding motif C of the riboswitch in addition to the two motifs A and B found in our previous study, and provided occupancies of the different binding motifs for each complex stoichiometry. Binding of a fourth neomycin B molecule was unspecific according to both MS and NMR data. Intriguingly, all major changes in the aptamer structure can be induced by the binding of the first neomycin B molecule regardless of whether it binds to motif A or B as evidenced by stoichiometry-resolved MS data together with titration data from 1H NMR spectroscopy in the imino proton region. Specific binding of the second and third neomycin B molecules further stabilizes the riboswitch aptamer, thereby allowing for a gradual response to increasing concentrations of neomycin B, which likely leads to a fine-tuning of the cellular regulatory mechanism.
Assuntos
Aptâmeros de Nucleotídeos , Framicetina , Riboswitch , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/genética , Sítios de Ligação , Framicetina/química , Framicetina/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Espectrometria de Massas/métodos , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Motivos de NucleotídeosRESUMO
Pathogenesis-related class 10 (PR-10) proteins play a crucial role in plant defense by acting as ribonucleases. The specific mechanism of action and substrate specificity of these proteins have remained largely unexplored so far. In this study, we elucidate the enzymatic activity of Pruâ p 1, a PR-10 protein from peach. We demonstrate that this protein catalyzes the endonucleolytic backbone cleavage of RNA substrates into short oligonucleotides. Initial cleavage products, identified through kinetic analysis, can bind again, priming them for further degradation. NMR binding site mapping reveals that the large internal cavity of Pruâ p 1, which is characteristic for PR-10 proteins, serves as an anchoring site for single-stranded ribonucleotide chains. We propose a structure-based mechanistic model that accounts for the observed cleavage patterns and the inhibitory effect of zeatin, a nucleoside analog, on the ribonuclease activity of Pruâ p 1.
Assuntos
Proteínas de Plantas , Sítios de Ligação , Endonucleases/metabolismo , Endonucleases/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , RNA/metabolismo , RNA/química , Especificidade por Substrato , Prunus persicaRESUMO
Understanding how ligands bind to ribonucleic acids (RNA) is important for understanding RNA recognition in biological processes and drug development. Here, we have studied neomycin B binding to neomycin-sensing riboswitch aptamer constructs by native top-down mass spectrometry (MS) using electrospray ionization (ESI) and collisionally activated dissociation (CAD). Our MS data for a 27 nt aptamer construct reveal the binding site and ligand interactions, in excellent agreement with the structure derived from nuclear magnetic resonance (NMR) studies. Strikingly, for an extended 40 nt aptamer construct, which represents the sequence with the highest regulatory factor for riboswitch function, we identified two binding motifs for neomycin B binding, one corresponding to the bulge-loop motif of the 27 nt construct and the other one in the minor groove of the lower stem, which according to the MS data are equally populated. By replacing a noncanonical with a canonical base pair in the lower stem of the 40 nt aptamer, we can reduce binding to the minor groove motif from â¼50 to â¼30%. Conversely, the introduction of a CUG/CUG motif in the lower stem shifts the binding equilibrium in favor of minor groove binding. The MS data reveal site-specific and stoichiometry-resolved information on aminoglycoside binding to RNA that is not directly accessible by other methods and underscore the role of noncanonical base pairs in RNA recognition by aminoglycosides.
Assuntos
Neomicina , Riboswitch , Framicetina , Antibacterianos/metabolismo , Aminoglicosídeos , RNA , Espectrometria de Massas , Sítios de Ligação , Conformação de Ácido Nucleico , LigantesRESUMO
The ongoing search for small molecule drugs that target ribonucleic acids (RNA) is complicated by a limited understanding of the principles that govern RNA-small molecule interactions. Here we have used stoichiometry-resolved native top-down mass spectrometry (MS) to study the binding of neomycin B to small model hairpin RNAs, an unstructured RNA, and a viral RNA construct. For 15-22â nt model RNAs with hairpin structure, we found that neomycin B binding to hairpin loops relies on interactions with both the nucleobases and the 2'-OH groups, and that a simple 5' or 3' overhang can introduce an additional binding motif. For a 47â nt RNA construct derived from stem IA of the human immunodeficiency virus 1 (HIV-1) rev response element (RRE) RNA, native top-down MS identified four different binding motifs, of which the purine-rich internal loop showed the highest affinity for neomycin B. Stoichiometry-resolved binding site mapping by native top-down MS allows for a new perspective on binding specificity, and has the potential to reveal unexpected principles of small molecule binding to RNA.
RESUMO
Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.