Structure and Internal Dynamics of Short RNA Duplexes Determined by a Combination of Pulsed EPR Methods and MD Simulations.

We used EPR spectroscopy to characterize the structure of RNA duplexes and their internal twist, stretch and bending motions. We prepared eight 20-base-pair-long RNA duplexes containing the rigid spin-label Çm, a cytidine analogue, at two positions and acquired orientation-selective PELDOR/DEER data. By using different frequency bands (X-, Q-, G-band), detailed information about the distance and orientation of the labels was obtained and provided insights into the global conformational dynamics of the RNA duplex. We used 19F Mims ENDOR experiments on three singly Çm- and singly fluorine-labeled RNA duplexes to determine the exact position of the Çm spin label in the helix. In a quantitative comparison to MD simulations of RNA with and without Çm spin labels, we found that state-of-the-art force fields with explicit parameterization of the spin label were able to describe the conformational ensemble present in our experiments. The MD simulations further confirmed that the Çm spin labels are excellent mimics of cytidine inducing only small local changes in the RNA structure. Çm spin labels are thus ideally suited for high-precision EPR experiments to probe the structure and, in conjunction with MD simulations, motions of RNA.

Hierarchical Assembly of Single-Stranded RNA.

Single-stranded RNA (ssRNA) plays a major role in the flow of genetic information-most notably, in the form of messenger RNA (mRNA)-and in the regulation of biological processes. The highly dynamic nature of chains of unpaired nucleobases challenges structural characterizations of ssRNA by experiments or molecular dynamics (MD) simulations alike. Here, we use hierarchical chain growth (HCG) to construct ensembles of ssRNA chains. HCG assembles the structures of protein and nucleic acid chains from fragment libraries created by MD simulations. Applied to homo- and heteropolymeric ssRNAs of different lengths, we find that HCG produces structural ensembles that overall are in good agreement with diverse experiments, including nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET). The agreement can be further improved by ensemble refinement using Bayesian inference of ensembles (BioEn). HCG can also be used to assemble RNA structures that combine base-paired and base-unpaired regions, as illustrated for the 5' untranslated region (UTR) of SARS-CoV-2 RNA.

Liquid-Liquid Phase Separation of the Intrinsically Disordered Domain of the Fused in Sarcoma Protein Results in Substantial Slowing of Hydration Dynamics.

Formation of liquid condensates plays a critical role in biology via localization of different components or via altered hydrodynamic transport, yet the hydrogen-bonding environment within condensates, pivotal for solvation, has remained elusive. We explore the hydrogen-bond dynamics within condensates formed by the low-complexity domain of the fused in sarcoma protein. Probing the hydrogen-bond dynamics sensed by condensate proteins using two-dimensional infrared spectroscopy of the protein amide I vibrations, we find that frequency-frequency correlations of the amide I vibration decay on a picosecond time scale. Interestingly, these dynamics are markedly slower for proteins in the condensate than in a homogeneous protein solution, indicative of different hydration dynamics. All-atom molecular dynamics simulations confirm that lifetimes of hydrogen-bonds between water and the protein are longer in the condensates than in the protein in solution. Altered hydrogen-bonding dynamics may contribute to unique solvation and reaction dynamics in such condensates.

Driving forces behind phase separation of the carboxy-terminal domain of RNA polymerase II.

Eukaryotic gene regulation and pre-mRNA transcription depend on the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II. Due to its highly repetitive, intrinsically disordered sequence, the CTD enables clustering and phase separation of Pol II. The molecular interactions that drive CTD phase separation and Pol II clustering are unclear. Here, we show that multivalent interactions involving tyrosine impart temperature- and concentration-dependent self-coacervation of the CTD. NMR spectroscopy, molecular ensemble calculations and all-atom molecular dynamics simulations demonstrate the presence of diverse tyrosine-engaging interactions, including tyrosine-proline contacts, in condensed states of human CTD and other low-complexity proteins. We further show that the network of multivalent interactions involving tyrosine is responsible for the co-recruitment of the human Mediator complex and CTD during phase separation. Our work advances the understanding of the driving forces of CTD phase separation and thus provides the basis to better understand CTD-mediated Pol II clustering in eukaryotic gene transcription.

Structural basis of substrate recognition by human tRNA splicing endonuclease TSEN.

Heterotetrameric human transfer RNA (tRNA) splicing endonuclease TSEN catalyzes intron excision from precursor tRNAs (pre-tRNAs), utilizing two composite active sites. Mutations in TSEN and its associated RNA kinase CLP1 are linked to the neurodegenerative disease pontocerebellar hypoplasia (PCH). Despite the essential function of TSEN, the three-dimensional assembly of TSEN-CLP1, the mechanism of substrate recognition, and the structural consequences of disease mutations are not understood in molecular detail. Here, we present single-particle cryogenic electron microscopy reconstructions of human TSEN with intron-containing pre-tRNAs. TSEN recognizes the body of pre-tRNAs and pre-positions the 3' splice site for cleavage by an intricate protein-RNA interaction network. TSEN subunits exhibit large unstructured regions flexibly tethering CLP1. Disease mutations localize far from the substrate-binding interface and destabilize TSEN. Our work delineates molecular principles of pre-tRNA recognition and cleavage by human TSEN and rationalizes mutations associated with PCH.

Structural ensembles of disordered proteins from hierarchical chain growth and simulation.

Disordered proteins and nucleic acids play key roles in cellular function and disease. Here, we review recent advances in the computational exploration of the conformational dynamics of flexible biomolecules. While atomistic molecular dynamics (MD) simulation has seen a lot of improvement in recent years, large-scale computing resources and careful validation are required to simulate full-length disordered biopolymers in solution. As a computationally efficient alternative, hierarchical chain growth (HCG) combines pre-sampled chain fragments in a statistically reproducible manner into ensembles of full-length atomically detailed biomolecular structures. Experimental data can be integrated during and after chain assembly. Applications to the neurodegeneration-linked proteins α-synuclein, tau, and TDP-43, including as condensate, illustrate the use of HCG. We conclude by highlighting the emerging connections to AI-based structural modeling including AlphaFold2.

Single-chain and condensed-state behavior of hnRNPA1 from molecular simulations.

Intrinsically disordered proteins (IDPs) are essential components for the formation of membraneless organelles, which play key functional and regulatory roles within biological systems. These complex assemblies form and dissolve spontaneously over time via liquid-liquid phase separation of IDPs. Mutations in their amino acid sequence can alter their phase behavior, which has been linked to the emergence of severe diseases. We study the conformation and phase behavior of a low-complexity domain of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) using coarse-grained implicit solvent molecular dynamics simulations. We systematically analyze how these properties are affected by the number of aromatic residues within the examined sequences. We find a significant compaction of the chains and an increase in the critical temperature with an increasing number of aromatic residues. The local persistence length is determined in single-chain simulations, revealing strong sequence-specific variations along the chain contour. Comparing single-chain and condensed-state simulations, we find many more collapsed polymer conformations in the dilute systems, even at temperatures near the estimated θ-temperature of the solution. These observations strongly support the hypothesis that aromatic residues play a dominant role in condensation, which is further corroborated by a detailed analysis of the intermolecular contacts, and conversely that important properties of condensates are captured in coarse-grained simulations. Interestingly, we observe density inhomogeneities within the condensates near criticality, which are driven by electrostatic interactions. Finally, we find that the relatively small fraction of hydrophobic residues in the IDPs results in interfacial tensions, which are significantly lower compared to typical combinations of immiscible simple liquids.

Substrate spectrum of PPM1D in the cellular response to DNA double-strand breaks.

PPM1D is a p53-regulated protein phosphatase that modulates the DNA damage response (DDR) and is frequently altered in cancer. Here, we employed chemical inhibition of PPM1D and quantitative mass spectrometry-based phosphoproteomics to identify the substrates of PPM1D upon induction of DNA double-strand breaks (DSBs) by etoposide. We identified 73 putative PPM1D substrates that are involved in DNA repair, regulation of transcription, and RNA processing. One-third of DSB-induced S/TQ phosphorylation sites are dephosphorylated by PPM1D, demonstrating that PPM1D only partially counteracts ATM/ATR/DNA-PK signaling. PPM1D-targeted phosphorylation sites are found in a specific amino acid sequence motif that is characterized by glutamic acid residues, high intrinsic disorder, and poor evolutionary conservation. We identified a functionally uncharacterized protein Kanadaptin as ATM and PPM1D substrate upon DSB induction. We propose that PPM1D plays a role during the response to DSBs by regulating the phosphorylation of DNA- and RNA-binding proteins in intrinsically disordered regions.

Transition Rates and Efficiency of Collective Variables from Time-Dependent Biased Simulations.

Simulations with adaptive time-dependent bias enable an efficient exploration of the conformational space of a system. However, the dynamic information is altered by the bias. Infrequent metadynamics recovers the transition rate of crossing a barrier, if the collective variables are ideal and there is no bias deposition near the transition state. Unfortunately, these conditions are not always fulfilled. To overcome these limitations, and inspired by single-molecule force spectroscopy, we use Kramers' theory for calculating the barrier-crossing rate when a time-dependent bias is added to the system. We assess the efficiency of collective variables parameter by measuring how efficiently the bias accelerates the transitions. We present approximate analytical expressions of the survival probability, reproducing the barrier-crossing time statistics and enabling the extraction of the unbiased transition rate even for challenging cases. We explore the limits of our method and provide convergence criteria to assess its validity.

Global Structure of the Intrinsically Disordered Protein Tau Emerges from Its Local Structure.

The paradigmatic disordered protein tau plays an important role in neuronal function and neurodegenerative diseases. To disentangle the factors controlling the balance between functional and disease-associated conformational states, we build a structural ensemble of the tau K18 fragment containing the four pseudorepeat domains involved in both microtubule binding and amyloid fibril formation. We assemble 129-residue-long tau K18 chains with atomic detail from an extensive fragment library constructed with molecular dynamics simulations. We introduce a reweighted hierarchical chain growth (RHCG) algorithm that integrates experimental data reporting on the local structure into the assembly process in a systematic manner. By combining Bayesian ensemble refinement with importance sampling, we obtain well-defined ensembles and overcome the problem of exponentially varying weights in the integrative modeling of long-chain polymeric molecules. The resulting tau K18 ensembles capture nuclear magnetic resonance (NMR) chemical shift and J-coupling measurements. Without further fitting, we achieve very good agreement with measurements of NMR residual dipolar couplings. The good agreement with experimental measures of global structure such as single-molecule Förster resonance energy transfer (FRET) efficiencies is improved further by ensemble refinement. By comparing wild-type and mutant ensembles, we show that pathogenic single-point P301L, P301S, and P301T mutations shift the population from the turn-like conformations of the functional microtubule-bound state to the extended conformations of disease-associated tau fibrils. RHCG thus provides us with an atomically detailed view of the population equilibrium between functional and aggregation-prone states of tau K18, and demonstrates that global structural characteristics of this intrinsically disordered protein emerge from its local structure.

Disease-linked TDP-43 hyperphosphorylation suppresses TDP-43 condensation and aggregation.

Post-translational modifications (PTMs) have emerged as key modulators of protein phase separation and have been linked to protein aggregation in neurodegenerative disorders. The major aggregating protein in amyotrophic lateral sclerosis and frontotemporal dementia, the RNA-binding protein TAR DNA-binding protein (TDP-43), is hyperphosphorylated in disease on several C-terminal serine residues, a process generally believed to promote TDP-43 aggregation. Here, we however find that Casein kinase 1δ-mediated TDP-43 hyperphosphorylation or C-terminal phosphomimetic mutations reduce TDP-43 phase separation and aggregation, and instead render TDP-43 condensates more liquid-like and dynamic. Multi-scale molecular dynamics simulations reveal reduced homotypic interactions of TDP-43 low-complexity domains through enhanced solvation of phosphomimetic residues. Cellular experiments show that phosphomimetic substitutions do not affect nuclear import or RNA regulatory functions of TDP-43, but suppress accumulation of TDP-43 in membrane-less organelles and promote its solubility in neurons. We speculate that TDP-43 hyperphosphorylation may be a protective cellular response to counteract TDP-43 aggregation.

Simulation of FUS Protein Condensates with an Adapted Coarse-Grained Model.

Disordered proteins and nucleic acids can condense into droplets that resemble the membraneless organelles observed in living cells. MD simulations offer a unique tool to characterize the molecular interactions governing the formation of these biomolecular condensates, their physicochemical properties, and the factors controlling their composition and size. However, biopolymer condensation depends sensitively on the balance between different energetic and entropic contributions. Here, we develop a general strategy to fine-tune the potential energy function for molecular dynamics simulations of biopolymer phase separation. We rebalance protein-protein interactions against solvation and entropic contributions to match the excess free energy of transferring proteins between dilute solution and condensate. We illustrate this formalism by simulating liquid droplet formation of the FUS low-complexity domain (LCD) with a rebalanced MARTINI model. By scaling the strength of the nonbonded interactions in the coarse-grained MARTINI potential energy function, we map out a phase diagram in the plane of protein concentration and interaction strength. Above a critical scaling factor of αc ≈ 0.6, FUS-LCD condensation is observed, where α = 1 and 0 correspond to full and repulsive interactions in the MARTINI model. For a scaling factor α = 0.65, we recover experimental densities of the dilute and dense phases, and thus the excess protein transfer free energy into the droplet and the saturation concentration where FUS-LCD condenses. In the region of phase separation, we simulate FUS-LCD droplets of four different sizes in stable equilibrium with the dilute phase and slabs of condensed FUS-LCD for tens of microseconds, and over one millisecond in aggregate. We determine surface tensions in the range of 0.01-0.4 mN/m from the fluctuations of the droplet shape and from the capillary-wave-like broadening of the interface between the two phases. From the dynamics of the protein end-to-end distance, we estimate shear viscosities from 0.001 to 0.02 Pa s for the FUS-LCD droplets with scaling factors α in the range of 0.625-0.75, where we observe liquid droplets. Significant hydration of the interior of the droplets keeps the proteins mobile and the droplets fluid.

Compaction of RNA Duplexes in the Cell*.

The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus laevis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA semi-quantitatively capture the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.

Local frustration determines loop opening during the catalytic cycle of an oxidoreductase.

Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein-binding sites. Here, we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point toward an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.

Hierarchical Ensembles of Intrinsically Disordered Proteins at Atomic Resolution in Molecular Dynamics Simulations.

Intrinsically disordered proteins (IDPs) constitute a large fraction of the human proteome and are critical in the regulation of cellular processes. A detailed understanding of the conformational dynamics of IDPs could help to elucidate their roles in health and disease. However, the inherent flexibility of IDPs makes structural studies and their interpretation challenging. Molecular dynamics (MD) simulations could address this challenge in principle, but inaccuracies in the simulation models and the need for long simulations have stymied progress. To overcome these limitations, we adopt a hierarchical approach that builds on the "flexible-meccano" model reported by Bernadó et al. (J. Am. Chem. Soc. 2005, 127, 17968-17969). First, we exhaustively sample small IDP fragments in all-atom simulations to capture their local structures. Then, we assemble the fragments into full-length IDPs to explore the stereochemically possible global structures of IDPs. The resulting ensembles of three-dimensional structures of full-length IDPs are highly diverse, much more so than in standard MD simulation. For the paradigmatic IDP α-synuclein, our ensemble captures both the local structure, as probed by nuclear magnetic resonance spectroscopy, and its overall dimension, as obtained from small-angle X-ray scattering in solution. By generating representative and meaningful starting ensembles, we can begin to exploit the massive parallelism afforded by current and future high-performance computing resources for atomic-resolution characterization of IDPs.

High-resolution EPR distance measurements on RNA and DNA with the non-covalent Ç´ spin label.

Pulsed electron paramagnetic resonance (EPR) experiments, among them most prominently pulsed electron-electron double resonance experiments (PELDOR/DEER), resolve the conformational dynamics of nucleic acids with high resolution. The wide application of these powerful experiments is limited by the synthetic complexity of some of the best-performing spin labels. The recently developed $\bf\acute{G}$ (G-spin) label, an isoindoline-nitroxide derivative of guanine, can be incorporated non-covalently into DNA and RNA duplexes via Watson-Crick base pairing in an abasic site. We used PELDOR and molecular dynamics (MD) simulations to characterize $\bf\acute{G}$, obtaining excellent agreement between experiments and time traces calculated from MD simulations of RNA and DNA double helices with explicitly modeled $\bf\acute{G}$ bound in two abasic sites. The MD simulations reveal stable hydrogen bonds between the spin labels and the paired cytosines. The abasic sites do not significantly perturb the helical structure. $\bf\acute{G}$ remains rigidly bound to helical RNA and DNA. The distance distributions between the two bound $\bf\acute{G}$ labels are not substantially broadened by spin-label motions in the abasic site and agree well between experiment and MD. $\bf\acute{G}$ and similar non-covalently attached spin labels promise high-quality distance and orientation information, also of complexes of nucleic acids and proteins.

Efficient Ensemble Refinement by Reweighting.

Ensemble refinement produces structural ensembles of flexible and dynamic biomolecules by integrating experimental data and molecular simulations. Here we present two efficient numerical methods to solve the computationally challenging maximum-entropy problem arising from a Bayesian formulation of ensemble refinement. Recasting the resulting constrained weight optimization problem into an unconstrained form enables the use of gradient-based algorithms. In two complementary formulations that differ in their dimensionality, we optimize either the log-weights directly or the generalized forces appearing in the explicit analytical form of the solution. We first demonstrate the robustness, accuracy, and efficiency of the two methods using synthetic data. We then use NMR J-couplings to reweight an all-atom molecular dynamics simulation ensemble of the disordered peptide Ala-5 simulated with the AMBER99SB*-ildn-q force field. After reweighting, we find a consistent increase in the population of the polyproline-II conformations and a decrease of α-helical-like conformations. Ensemble refinement makes it possible to infer detailed structural models for biomolecules exhibiting significant dynamics, such as intrinsically disordered proteins, by combining input from experiment and simulation in a balanced manner.

Dispersion Correction Alleviates Dye Stacking of Single-Stranded DNA and RNA in Simulations of Single-Molecule Fluorescence Experiments.

We combine single-molecule Förster resonance energy transfer (single-molecule FRET) experiments with extensive all-atom molecular dynamics (MD) simulations (>100 µs) to characterize the conformational ensembles of single-stranded (ss) DNA and RNA in solution. From MD simulations with explicit dyes attached to single-stranded nucleic acids via flexible linkers, we calculate FRET efficiencies and fluorescence anisotropy decays. We find that dispersion-corrected water models alleviate the problem of overly abundant interactions between fluorescent dyes and the aromatic ring systems of nucleobases. To model dye motions in a computationally efficient and conformationally exhaustive manner, we introduce a dye-conformer library, built from simulations of dinucleotides with covalently attached dye molecules. We use this library to calculate FRET efficiencies for dT19, dA19, and rA19 simulated without explicit labels over a wide range of salt concentrations. For end-labeled homopolymeric pyrimidine ssDNA, MD simulations with the parmBSC1 force field capture the overall trend in salt-dependence of single-molecule FRET based distance measurements. For homopolymeric purine ssRNA and ssDNA, the DESRES and parmBSC1 force fields, respectively, provide useful starting points, even though our comparison also identifies clear deviations from experiment.

Optimal Destabilization of DNA Double Strands by Single-Nucleobase Caging.

Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF-amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu-catalyzed azide-alkyne cycloaddition post-synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o-nitrobenzyl-caged (NPBY-) and diethylaminocoumarin-cages (DEACM-) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base-pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri-substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove.

Precision DEER Distances from Spin-Label Ensemble Refinement.

Double electron-electron resonance (DEER) experiments probe nanometer-scale distances in spin-labeled proteins and nucleic acids. Rotamer libraries of the covalently attached spin-labels help reduce position uncertainties. Here we show that rotamer reweighting is essential for precision distance measurements, making it possible to resolve Ångstrom-scale domain motions. We analyze extensive DEER measurements on the three N-terminal polypeptide transport-associated (POTRA) domains of the outer membrane protein Omp85. Using the "Bayesian inference of ensembles" maximum-entropy method, we extract rotamer weights from the DEER measurements. Small weight changes suffice to eliminate otherwise significant discrepancies between experiments and model and unmask 1-3 Å domain motions relative to the crystal structure. Rotamer-weight refinement is a simple yet powerful tool for precision distance measurements that should be broadly applicable to label-based measurements including DEER, paramagnetic relaxation enhancement, and fluorescence resonance energy transfer (FRET).