A generalized essential dynamics-refined ENM (edENM) is introduced for both DNA, RNA, and protein-nucleic acid complexes, parametrized against a diverse set of molecular dynamics simulations and validated using experimental ensembles from nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy.
Abstract
Abstract The flexibility of nucleic acids plays a central role in numerous biological processes, including chromatin organization, gene regulation, and ribosome assembly. While elastic network models (ENMs) have successfully captured conformational changes in proteins through harmonic normal modes (NMs), analogous approaches for nucleic acids remain limited. Here, we introduce a generalized essential dynamics-refined ENM (edENM) for both DNA, RNA, and protein–nucleic acid complexes, parametrized against a diverse set of molecular dynamics simulations and validated using experimental ensembles from nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy. edENM achieves high agreement with experimental conformational changes across a curated benchmark of ∼60 DNA, RNA, and protein–nucleic acid systems. Compared to uniform-spring parametrizations, it produces significantly more collective NMs and suppresses unphysical backbone ruptures. We further integrate edENM into eBDIMS2, an efficient Brownian Dynamics path-sampling framework, extending its applicability to nucleic acid-containing systems at the megadalton scale. This enables the exploration of complex conformational transitions, including rearrangements of RNA folds in coronaviruses, large-scale remodeling in Argonaute–RNA complexes, multi-nucleosome assemblies in chromatin, as well as ribosomal particles. Together, these results establish an accessible and scalable elastic network framework for modeling conformational changes across the full spectrum of nucleic acid-containing biological systems.
Biomolecular condensates function as membraneless compartments, and some protein condensates can selectively concentrate single-stranded nucleic acids while excluding double-stranded nucleic acids. Understanding how nucleic acid structure affects partitioning into condensates has important implications for nucleic acid activity and function within condensates. Here, we present a set of coarse-grained two-bead-per-nucleotide models for simulations of double-stranded RNA and DNA in the CALVADOS framework. Our models separately represent the backbone and base, and maintain the helical structures using an elastic network potential tuned to capture chain stiffness. For dsRNA, the base stickiness was tuned using experimental data on differential partitioning of single- and double-stranded RNA into Ddx4N1 condensates in order to account for reduced base accessibility upon duplex formation. This RNA structural selectivity varied with the balance of electrostatic and non-electrostatic interactions, as revealed by simulations of condensates of the CAPRIN1 disordered region at varying ionic concentrations and with an R-to-K sequence variant. Finally, we developed parameters for double-stranded DNA using a similar approach. We envision that the CALVADOS models for double-stranded RNA and DNA will be useful for studying co-condensates of proteins and structured nucleic acids.
Ikki Yasuda, G. Tesei, Eiji Yamamoto et al.· bioRxiv· 0 citations
Proteins are dynamic molecular machines that change shape in response to physical and chemical perturbations. Although single-molecule force spectroscopy provides precise information about the stretching of proteins in response to tunable forces, it does so without structural detail. Circular protein-DNA chimeras, with DNA attached to pairs of surface sites, have been introduced as an alternative way to tunably apply forces to proteins. Intriguingly, these chimeras should be tractable for atomic-level study by nuclear magnetic resonance (NMR) spectroscopy and other structural methods. Here, we describe the NMR-scale synthesis of circular chimeras of single-and double-stranded DNA with ubiquitin, an essential component of many cellular pathways. We designed these chimeras to probe a two-residue retraction of ubiquitin’s C-terminal β5 strand, normally triggered by phosphorylation of serine 65 during initiation of mitophagy. We probed the resulting conformational changes by NMR and found that the attachment of a single strand of DNA suffices to alter this conformational equilibrium. A control bearing two separate short single DNA strands recapitulated much of the circular chimera’s NMR properties, supporting a dominant role for local protein-DNA interactions rather than spring-like action by single- or double-stranded DNA. These results provide a necessary benchmark for future studies using DNA springs to probe the functional dynamics of proteins. Significance statement Ligands, post-translational modifications, and mechanical inputs reshape proteins through forces that propagate across their structures. The underlying mechanical properties of proteins mediating these changes are rarely accessible with atomic-level detail. By producing NMR-scale circular protein-DNA chimeras, we provide a route to examining how defined physical perturbations alter protein conformational land-scapes. The results establish both the promise of this strategy and the need to account for local DNA-protein interactions.
S. Boral, Michael D. Schnebly, D. Gamada et al.· bioRxiv· 0 citations
Molecular dynamics simulations of nucleic acids are performed using a solvent-buffer distance of 10 Å between the solute surface and the simulation box boundary. Although this cell size has been extensively explored in protein simulations, its implications for nucleic acid dynamics are not well understood. Nucleic acids are elongated, highly charged, and flexible structures with hydration and dynamical properties distinct from those of proteins and therefore, they may require different solvent-layer considerations in simulations. In this study, we investigated the effect of simulation cell size on nucleic acid dynamics by simulating a 30-base-pair double-helical nucleic acid structure and its two single-stranded forms using solvent-buffer distances of 3, 5, 10, 15, and 20 Å. Smaller cells may impose restricted hydration, molecular crowding, and periodic image interactions. However, larger cells provide solvent space for conformational relaxation. A total of 45 µs of molecular dynamics simulations were performed (3 structures × 5 cell sizes × 3 replicates × 1 µs). Our results show that while the commonly used 10 Å buffer may be sufficient to maintain the stability of the double-stranded nucleic acid, larger cells are required to capture the conformational dynamics of single-stranded structures. In both, increasing the cell size to 15 or 20 Å enables broader conformational sampling. The first hydration shell exhibits reduced crowding in the 20 Å cell, consistent with more relaxed conformations. At larger cell sizes, single-stranded nucleic acids adopt compact, self-associated conformations for stability. Together, this study presents physical insight into how simulation cell size and solvent environment influence nucleic acid dynamics.
Nainsy Baghel, Pranchal Shrivastava, R. Mehra· bioRxiv· 0 citations
Protein-RNA complexes underlie essential cellular processes and understanding their functional mechanisms requires structural analysis. Yet, their inherent flexibility, multi-valency, and dynamics make them challenging targets for structural biology. Integrative approaches, combining nuclear magnetic resonance (NMR), small-angle scattering, cryo-electron microscopy (cryo-EM), cryo-electron tomography (cryo-ET), crosslinking mass spectrometry, single-molecule techniques, and artificial intelligence (AI)-based predictions, have enabled the characterization of increasingly complex ribonucleoprotein (RNP) assemblies, in vitro and in their cellular contexts. These strategies have begun to capture molecular architecture and dynamic behaviors across time and space, paving the way for 4D structural biology. Here, we review recent developments in integrative modeling of protein-RNA complexes, highlighting advances in in-cell, 4D and condensate structural biology, and discuss how these approaches shape our understanding of RNP assembly, regulation, and function in physiologically relevant environments.
Simone Heber, Janosch Hennig· Current Opinion in Structura...· 1 citation
This work assesses ML potentials for exploring RNA conformations using the adenine–adenine dinucleoside monophosphate (ApA) dimer, a fundamental RNA building block, and parametrized ML potentials based on the equivariant MACE architecture and informed by both ab initio and semiempirical property data.
Leonardo Medrano Sandonas, Macarena Tolmos Nehme, L. F. Cofas-Vargas et al.· Journal of Chemical Theory a...· 0 citations
Physical remodeling of chromatin by non-histone architectural proteins of the High Mobility Group B (HMGB) family is central to eukaryotic transcriptional regulation. Nhp6A, the prototypical single-HMG-box protein from yeast, harbors both ordered and disordered regions enabling it to bind and bend DNA without sequence specificity. Here, we integrate ensemble experiments, single-molecule FRET, statistical mechanical modeling and atomistic simulations to dissect the structural and functional consequences of context-dependent phosphorylation in the ordered domain and its interplay with the intrinsically disordered region in Nhp6A. We find that Nhp6A occupies a narrow thermodynamic window, with a melting temperature close to the growth temperature of its host organism and high unfolding cooperativity, a feature conserved across the HMG-box family. Phosphorylation extents – mimicked by multisite phosphomimetic substitutions at residue positions conserved across fungal taxa – smoothly tuning the conformational equilibria between at least two different substates in the native ensemble, apart from the unfolded state. This intrinsic plasticity enables close packing of Nhp6A on DNA through two degenerate binding modes, accompanied by two distinct DNA bending geometries. DNA rescues a strongly destabilized mutant, T63D, through favorable intermolecular interactions, thus effectively acting as a chaperone driving folding. Our findings thus reveal a conserved sequence-ensemble-dynamics code in Nhp6A wherein not just stability, but also phosphorylation-induced conformational switching, disordered tail dynamics, and DNA binding-bending closely coordinate chromatin accessibility. The combination of marginal stability, large cooperativity and electrostatic frustration emerges as a design principle to encode charge sensitivity into proteins, and may represent a general strategy for multisite post-translational regulation.
Shilpi Laha, H. Madhan, Yuji Itoh et al.· bioRxiv· 0 citations
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