Determining structures of individual RNA conformers using atomic force microscopy images and deep neural networks.

The vast percentage of the human genome is transcribed into RNA, many of which contain various structural elements and are important for functions. RNA molecules are conformationally heterogeneous and functionally dyanmics1, even when they are structured and well-folded2, which limit the applicability of methods such as NMR, crystallography, or cryo-EM. Moreover, because of the lack of a large structure RNA database, and no clear correlation between sequence and structure, approaches like AlphaFold3 for protein structure prediction, do not apply to RNA. Therefore determining the structures of heterogeneous RNA is an unmet challenge. Here we report a novel method of determining RNA three-dimensional topological structures using deep neural networks and atomic force microscopy (AFM) images of individual RNA molecules in solution. Owing to the high signal-to-noise ratio of AFM, our method is ideal for capturing structures of individual conformationally heterogeneous RNA. We show that our method can determine 3D topological structures of any large folded RNA conformers, from ~ 200 to ~ 420 residues, the size range that most functional RNA structures or structural elements fall into. Thus our method addresses one of the major challenges in frontier RNA structural biology and may impact our fundamental understanding of RNA structure.

Molecular Structure and Functional Analysis of Pyocin S8 from Pseudomonas aeruginosa Reveals the Essential Requirement of a Glutamate Residue in the H-N-H Motif for DNase Activity.

Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact.

CALX-CBD1 Ca2+-Binding Cooperativity Studied by NMR Spectroscopy and ITC with Bayesian Statistics.

The Na+/Ca2+ exchanger of Drosophila melanogaster, CALX, is the main Ca2+-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Na+/Ca2+ exchangers have two Ca2+ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2+ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2+ at physiological concentrations. CALX-CBD1 consists of a ß-sandwich and displays four Ca2+-binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2+ binding to CALX-CBD1. We observed that this domain binds Ca2+ in the slow exchange regime at the NMR chemical shift timescale. Ca2+ binding restricts the dynamics in the Ca2+-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2 dispersion allowed the determination of Ca2+ dissociation rates (∼30 s-1). NMR titration curves of residues in the Ca2+-binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2+-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill ∼2.9, near maximal cooperativity (nHill = 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2+ is approximately three orders of magnitude higher than that of binding a single Ca2+. Hence, four Ca2+ ions bind almost simultaneously to CALX-CBD1. Cooperative Ca2+ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2+ concentration in sensory neuronal cells.