RNA dynamics from experimental and computational approaches.

Conformational dynamics is crucial for the biological function of RNA molecules and for their potential as therapeutic targets. This meeting report outlines key "take-home" messages that emerged from the presentations and discussions during the CECAM workshop "RNA dynamics from experimental and computational approaches" in Paris, June 26-28, 2023.

SETD8 inhibition targets cancer cells with increased rates of ribosome biogenesis.

SETD8 is a methyltransferase that is overexpressed in several cancers, which monomethylates H4K20 as well as other non-histone targets such as PCNA or p53. We here report novel SETD8 inhibitors, which were discovered while trying to identify chemicals that prevent 53BP1 foci formation, an event mediated by H4K20 methylation. Consistent with previous reports, SETD8 inhibitors induce p53 expression, although they are equally toxic for p53 proficient or deficient cells. Thermal stability proteomics revealed that the compounds had a particular impact on nucleoli, which was confirmed by fluorescent and electron microscopy. Similarly, Setd8 deletion generated nucleolar stress and impaired ribosome biogenesis, supporting that this was an on-target effect of SETD8 inhibitors. Furthermore, a genome-wide CRISPR screen identified an enrichment of nucleolar factors among those modulating the toxicity of SETD8 inhibitors. Accordingly, the toxicity of SETD8 inhibition correlated with MYC or mTOR activity, key regulators of ribosome biogenesis. Together, our study provides a new class of SETD8 inhibitors and a novel biomarker to identify tumors most likely to respond to this therapy.

An integrated machine-learning model to predict nucleosome architecture.

We demonstrate that nucleosomes placed in the gene body can be accurately located from signal decay theory assuming two emitters located at the beginning and at the end of genes. These generated wave signals can be in phase (leading to well defined nucleosome arrays) or in antiphase (leading to fuzzy nucleosome architectures). We found that the first (+1) and the last (-last) nucleosomes are contiguous to regions signaled by transcription factor binding sites and unusual DNA physical properties that hinder nucleosome wrapping. Based on these analyses, we developed a method that combines Machine Learning and signal transmission theory able to predict the basal locations of the nucleosomes with an accuracy similar to that of experimental MNase-seq based methods.

Developments and applications of the OPTIMADE API for materials discovery, design, and data exchange.

The Open Databases Integration for Materials Design (OPTIMADE) application programming interface (API) empowers users with holistic access to a growing federation of databases, enhancing the accessibility and discoverability of materials and chemical data. Since the first release of the OPTIMADE specification (v1.0), the API has undergone significant development, leading to the v1.2 release, and has underpinned multiple scientific studies. In this work, we highlight the latest features of the API format, accompanying software tools, and provide an update on the implementation of OPTIMADE in contributing materials databases. We end by providing several use cases that demonstrate the utility of the OPTIMADE API in materials research that continue to drive its ongoing development.

Special issue: Multiscale simulations of DNA from electrons to nucleosomes.

This editorial for Volume 16, Issue 3 of Biophysical Reviews highlights the three-dimensional structural and dynamic information encoded in DNA sequences and introduces the topics covered in this special issue of the journal on Multiscale Simulations of DNA from Electrons to Nucleosomes. Biophysical Reviews is the official journal of the International Union for Pure and Applied Biophysics (IUPAB 2024). The international scope of the articles in the issue exemplifies the goals of IUPAB to organize worldwide advancements, co-operation, communication, and education in biophysics.

MD-DATA: the legacy of the ABC Consortium.

The ABC Consortium has been generating nucleic-acids MD trajectories for more than 20 years. This brief comment highlights the importance of this data for the field, which triggered a number of critical studies, including force-field parameterization and development of new coarse-grained and mesoscopic models. With the world entering into a new data-driven era led by artificial intelligence, where data is becoming more essential than ever, the ABC initiative is leading the way for nucleic acid flexibility.

Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Using interactive Jupyter Notebooks and BioConda for FAIR and reproducible biomolecular simulation workflows.

Interactive Jupyter Notebooks in combination with Conda environments can be used to generate FAIR (Findable, Accessible, Interoperable and Reusable/Reproducible) biomolecular simulation workflows. The interactive programming code accompanied by documentation and the possibility to inspect intermediate results with versatile graphical charts and data visualization is very helpful, especially in iterative processes, where parameters might be adjusted to a particular system of interest. This work presents a collection of FAIR notebooks covering various areas of the biomolecular simulation field, such as molecular dynamics (MD), protein-ligand docking, molecular checking/modeling, molecular interactions, and free energy perturbations. Workflows can be launched with myBinder or easily installed in a local system. The collection of notebooks aims to provide a compilation of demonstration workflows, and it is continuously updated and expanded with examples using new methodologies and tools.

CGeNArate: a sequence-dependent coarse-grained model of DNA for accurate atomistic MD simulations of kb-long duplexes.

We present CGeNArate, a new model for molecular dynamics simulations of very long segments of B-DNA in the context of biotechnological or chromatin studies. The developed method uses a coarse-grained Hamiltonian with trajectories that are back-mapped to the atomistic resolution level with extreme accuracy by means of Machine Learning Approaches. The method is sequence-dependent and reproduces very well not only local, but also global physical properties of DNA. The efficiency of the method allows us to recover with a reduced computational effort high-quality atomic-resolution ensembles of segments containing many kilobases of DNA, entering into the gene range or even the entire DNA of certain cellular organelles.

Nucleic acids in modern molecular therapies: A realm of opportunities for strategic drug design.

RNA vaccines have made evident to society what was already known by the scientific community: nucleic acids will be the "drugs of the future." By modifying the genome, interfering in transcription or translation, and by introducing new catalysts into the cell or by mimicking antibody effects, nucleic acids can generate therapeutic activities that are not accessible by any other therapeutic agents. There are, however, challenges that need to be solved in the next few years to make nucleic acids usable in a wide range of therapeutic scenarios. This review illustrates how simulation methods can help achieve this goal.

Main role of fractal-like nature of conformational space in subdiffusion in proteins.

Protein dynamics involves a myriad of mechanical movements happening at different time and space scales, which make it highly complex. One of the less understood features of protein dynamics is subdiffusivity, defined as sublinear dependence between displacement and time. Here, we use all-atoms molecular dynamics (MD) simulations to directly interrogate an already well-established theory and demonstrate that subdiffusivity arises from the fractal nature of the network of metastable conformations over which the dynamics, thought of as a diffusion process, takes place.

Molecular stripping underpins derepression of a toxin-antitoxin system.

Transcription factors control gene expression; among these, transcriptional repressors must liberate the promoter for derepression to occur. Toxin-antitoxin (TA) modules are bacterial elements that autoregulate their transcription by binding the promoter in a T:A ratio-dependent manner, known as conditional cooperativity. The molecular basis of how excess toxin triggers derepression has remained elusive, largely because monitoring the rearrangement of promoter-repressor complexes, which underpin derepression, is challenging. Here, we dissect the autoregulation of the Salmonella enterica tacAT3 module. Using a combination of assays targeting DNA binding and promoter activity, as well as structural characterization, we determine the essential TA and DNA elements required to control transcription, and we reconstitute a repression-to-derepression path. We demonstrate that excess toxin triggers molecular stripping of the repressor complex off the DNA through multiple allosteric changes causing DNA distortion and ultimately leading to derepression. Thus, our work provides important insight into the mechanisms underlying conditional cooperativity.

The Rhodium Analogue of Coenzyme B12 as an Anti-Photoregulatory Ligand Inhibiting Bacterial CarH Photoreceptors.

Coenzyme B12 (AdoCbl; 5'-deoxy-5'-adenosylcobalamin), the quintessential biological organometallic radical catalyst, has a formerly unanticipated, yet extensive, role in photoregulation in bacteria. The light-responsive cobalt-corrin AdoCbl performs this nonenzymatic role by facilitating the assembly of CarH photoreceptors into DNA-binding tetramers in the dark, suppressing gene expression. Conversely, exposure to light triggers the decomposition of this AdoCbl-bound complex by a still elusive photochemical mechanism, activating gene expression. Here, we have examined AdoRhbl, the non-natural rhodium analogue of AdoCbl, as a photostable isostructural surrogate for AdoCbl. We show that AdoRhbl closely emulates AdoCbl in its uptake by bacterial cells and structural functionality as a regulatory ligand for CarH tetramerization, DNA binding, and repressor activity. Remarkably, we find AdoRhbl is photostable even when bound "base-off/His-on" to CarH in vitro and in vivo. Thus, AdoRhbl, an antivitamin B12, also represents an unprecedented anti-photoregulatory ligand, opening a pathway to precisely target biomimetic inhibition of AdoCbl-based photoregulation, with new possibilities for selective antibacterial applications. Computational biomolecular analysis of AdoRhbl binding to CarH yields detailed structural insights into this complex, which suggest that the adenosyl group of photoexcited AdoCbl bound to CarH may specifically undergo a concerted non-radical syn-1,2-elimination mechanism, an aspect not previously considered for this photoreceptor.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.

A new paradigm for molecular dynamics databases: the COVID-19 database, the legacy of a titanic community effort.

Molecular dynamics (MD) simulations are keeping computers busy around the world, generating a huge amount of data that is typically not open to the scientific community. Pioneering efforts to ensure the safety and reusability of MD data have been based on the use of simple databases providing a limited set of standard analyses on single-short trajectories. Despite their value, these databases do not offer a true solution for the current community of MD users, who want a flexible analysis pipeline and the possibility to address huge non-Markovian ensembles of large systems. Here we present a new paradigm for MD databases, resilient to large systems and long trajectories, and designed to be compatible with modern MD simulations. The data are offered to the community through a web-based graphical user interface (GUI), implemented with state-of-the-art technology, which incorporates system-specific analysis designed by the trajectory providers. A REST API and associated Jupyter Notebooks are integrated into the platform, allowing fully customized meta-analysis by final users. The new technology is illustrated using a collection of trajectories obtained by the community in the context of the effort to fight the COVID-19 pandemic. The server is accessible at https://bioexcel-cv19.bsc.es/#/. It is free and open to all users and there are no login requirements. It is also integrated into the simulations section of the BioExcel-MolSSI COVID-19 Molecular Structure and Therapeutics Hub: https://covid.molssi.org/simulations/ and is part of the MDDB effort (https://mddbr.eu).

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α.

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

The pseudotorsional space of RNA.

The characterization of the conformational landscape of the RNA backbone is rather complex due to the ability of RNA to assume a large variety of conformations. These backbone conformations can be depicted by pseudotorsional angles linking RNA backbone atoms, from which Ramachandran-like plots can be built. We explore here different definitions of these pseudotorsional angles, finding that the most accurate ones are the traditional η (eta) and θ (theta) angles, which represent the relative position of RNA backbone atoms P and C4'. We explore the distribution of η - θ in known experimental structures, comparing the pseudotorsional space generated with structures determined exclusively by one experimental technique. We found that the complete picture only appears when combining data from different sources. The maps provide a quite comprehensive representation of the RNA accessible space, which can be used in RNA-structural predictions. Finally, our results highlight that protein interactions lead to significant changes in the population of the η - θ space, pointing toward the role of induced-fit mechanisms in protein-RNA recognition.

Sequence-Dependent Properties of the RNA Duplex.

Sequence-dependent properties of the DNA duplex have been accurately described using extensive molecular dynamics simulations. The RNA duplex meanwhile─which is typically represented as a sequence-averaged rigid rod─does not benefit from having equivalent molecular dynamics simulations. In this paper, we present a massive simulation effort using a set of ABC-optimized duplexes from which we derived tetramer-resolution properties of the RNA duplex and a simple mesoscopic model that can represent elastic properties of long RNA duplexes. Despite the extreme chemical similarity between DNA and RNA, the local and global elastic properties of the duplexes are very different. DNA duplexes show a complex and nonelastic pattern of flexibility, for instance, while RNA duplexes behave as an elastic system whose deformations can be represented by simple harmonic potentials. In RNA duplexes (RNA2), not only are intra- and interbase pair parameters (equilibrium and mechanical) different from those in the equivalent DNA duplex sequences (DNA2) but the correlations between movements also differ. Simple statements on the relative flexibility or stability of both polymers are meaningless and should be substituted by a more detailed description depending on the sequence and the type of deformation considered.

Histone H3 serine-57 is a CHK1 substrate whose phosphorylation affects DNA repair.

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.

Molecular dynamics modelling of the interaction of a synthetic zinc-finger miniprotein with DNA.

We report the modelling of the DNA complex of an artificial miniprotein composed of two zinc finger modules and an AT-hook linking peptide. The computational study provides for the first time a structural view of these types of complexes, dissecting interactions that are key to modulate their stability. The relevance of these interactions was validated experimentally. These results confirm the potential of this type of computational approach for studying peptide-DNA complexes and suggest that they could be very useful for the rational design of non-natural, DNA binding miniproteins.