Biology subjects

Sponer, J.

Publications and source records attributed to Sponer, J..

33 records · Page 2Linked to original sources

bioRxiv2023

Complexity of Guanine Quadruplex Unfolding Pathways Revealed by Atomistic Pulling Simulations

Guanine quadruplexes (GQs) are non-canonical nucleic acid structures involved in many biological processes. GQs formed in single-stranded regions often need to be unwound by cellular machinery, so their mechanochemical properties are important. Here, we performed steered molecular dynamics simulations of human telomeric GQs to study their unfolding. We examined four pulling regimes, including very slow setup with pulling velocity and force load accessible to high-speed atomic force microscopy. We identified multiple factors affecting the unfolding mechanism. The more the direction of force was perpendicular to the GQ channel axis (determined by GQ topology), the more the base unzipping mechanism happened. If the GQ had either all-anti or all-syn pattern in a strand, strand slippage mechanism was more likely to occur. Importantly, slower pulling velocity led to richer unfolding pathways including partial refolding attempts. We show that GQ may eventually unfold after force drop under forces smaller than those the GQ withstood before the drop. This suggests that proteins in vivo might resolve GQs even if their stall forces are smaller than GQ rupture force. Finally, we found out that different unfolding intermediates may have very similar chain end-to-end distance, which reveals some limitations of structural interpretations of single-molecule spectroscopic data.

biophysics↗

bioRxiv2022

Conformational Heterogeneity of RNA Stem-Loop Hairpins Bound to FUS RNA Recognition Motif with Disordered RGG Tail Revealed by Unbiased Molecular Dynamics Simulations.

RNA-protein complexes use diverse binding strategies, ranging from structurally well-defined interfaces to completely disordered regions. Experimental characterization of flexible segments is challenging and can be aided by atomistic molecular dynamics (MD) simulations. Here we used extended set of microsecond-scale MD trajectories (400 s in total) to study two FUS-RNA constructs previously characterized by NMR spectroscopy. The FUS protein contains well-structured RNA Recognition Motif domain followed by presumably disordered RGG tail and bind RNA stem-loop hairpins. Our simulations provide several suggestions complementing the experiments but also reveal major methodological difficulties in studies of such complex RNA-protein interfaces. Despite efforts to stabilize the binding via system-specific force-field adjustments, we have observed progressive distortions of the RNA-protein interface inconsistent with experimental data, as in detail documented. We further propose that the dynamics is so rich that its converged description would not be achievable even upon stabilizing the system. Still, after careful analysis of the trajectories, we have made several suggestions regarding the binding. We identify substates in the RNA loops which can explain the NOE data. The RGG tail localized in the minor groove remains disordered, sampling countless transient interactions with the RNA. There are long-range couplings among the different elements contributing to the recognition, which can lead to allosteric communication throughout the system. Overall, the RNA-FUS systems form dynamical ensembles that cannot be fully represented by single static structures. Thus, albeit imperfect, MD simulations represent a viable tool to investigate them.

molecular biology↗

bioRxiv2022

Spontaneous binding of single-stranded RNAs to RRM proteins visualised by unbiased atomistic simulations with rescaled RNA force field

Recognition of single-stranded RNA (ssRNA) by RNA recognition motif (RRM) domains is an important class of protein-RNA interactions. Many such complexes were characterized using NMR and/or X-ray crystallography techniques, revealing ensemble-averaged pictures of the bound states. However, it is becoming widely accepted that better understanding of protein-RNA interactions would be obtained from ensemble descriptions. Indeed, earlier molecular dynamics (MD) simulations of bound states indicated visible dynamics at the RNA-RRM interfaces. Here, we report the first atomistic simulation study of spontaneous binding of short RNA sequences to RRM domains of HuR and SRSF1 proteins. Using millisecond-scale aggregate ensemble of unbiased simulations we were able to observe a few dozens of binding events. The HuR RRM3 utilizes a pre-binding state to navigate the RNA sequence to its partially disordered bound state and then to dynamically scan its different binding registers. The SRFS1 RRM2 binding is more straightforward but still multiple-pathway. The present study necessitated development of a goal-specific force-field modification scaling down the intramolecular vdW interactions of the RNA which also improves description of the RNA-RRM bound state. Our study opens a new avenue for large-scale atomistic investigations of binding landscapes of protein-RNA complexes and future perspectives of such research are discussed.

molecular biology↗

bioRxiv2021

Towards Convergence in Folding Simulations of RNA Tetraloops: Comparison of Enhanced Sampling Techniques and Effects of Force Field Corrections

Atomistic molecular dynamics (MD) simulations represent established technique for investigation of RNA structural dynamics. Despite continuous development, contemporary RNA simulations still suffer from suboptimal accuracy of empirical potentials (force fields, ffs) and sampling limitations. Development of efficient enhanced sampling techniques is important for two reasons. First, they allow to overcome the sampling limitations and, second, they can be used to quantify ff imbalances provided they reach a sufficient convergence. Here, we study two RNA tetraloops (TLs), namely the GAGA and UUCG motifs. We perform extensive folding simulations and calculate folding free energies ({Delta}Gfold) with the aim to compare different enhanced sampling techniques and to test several modifications of the nonbonded terms extending the AMBER OL3 RNA ff. We demonstrate that replica exchange solute tempering (REST2) simulations with 12-16 replicas do not show any sign of convergence even when extended to time scale of 120 s per replica. However, combination of REST2 with well-tempered metadynamics (ST-MetaD) achieves good convergence on a time-scale of 5-10 s per replica, improving the sampling efficiency by at least two orders of magnitude. Effects of ff modifications on {Delta}Gfold energies were initially explored by the reweighting approach and then validated by new simulations. We tested several manually-prepared variants of gHBfix potential which improve stability of the native state of both TLs by up to ~2 kcal/mol. This is sufficient to conveniently stabilize the folded GAGA TL while the UUCG TL still remains under-stabilized. Appropriate adjustment of van der Waals parameters for C-H...O5 base-phosphate interaction are also shown to be capable of further stabilizing the native states of both TLs by ~0.6 kcal/mol.

biophysics↗

bioRxiv2021

Z-DNA as a Touchstone for Additive Empirical Force Fields and a Refinement of the Alpha/Gamma DNA torsions for AMBER

Although current AMBER force fields are relatively accurate for canonical B-DNA, many non-canonical structures are still described incorrectly. As non-canonical motifs are attracting increasing attention due to the role they play in living organisms, further improvement is desirable. Here, we have chosen Z-DNA molecule, can be considered a touchstone of the universality of empirical force fields, since the non-canonical and {gamma} backbone conformations native to Z-DNA are also found in protein-DNA complexes, i-motif DNA and other non-canonical DNAs. We show that spurious /{gamma} conformations occurring in simulations with current AMBER force fields, OL15 and bsc1, are largely due to inaccurate /{gamma} parameterization. Moreover, stabilization of native Z-DNA substates involving {gamma} = trans conformations appears to be in conflict with the correct description of the canonical B-DNA structure. Because the balance of the native and spurious conformations is influenced by non-additive effects, this is a difficult case for an additive dihedral energy scheme such as AMBER. We propose new /{gamma} parameters, denoted OL21, and show that they improve the stability of native /{gamma} Z-DNA substates while keeping the canonical DNA description virtually unchanged, and thus represent a reasonable compromise within the additive force field framework. Although further extensive testing is needed, the new modification appears to be a promising step towards a more reliable description of non-canonical DNA motifs and provides the best performance for Z-DNA molecules among current AMBER force fields.

biophysics↗

bioRxiv2021

Recognition of N6-methyladenosine by the YTHDC1 YTH domain studied by molecular dynamics and NMR spectroscopy: The role of hydration

The YTH domain of YTHDC1 belongs to a class of protein "readers", recognizing the N6-methyladenosine (m6A) chemical modification in mRNA. Static ensemble-averaged structures revealed details of N6-methyl recognition via a conserved aromatic cage. Here, we performed molecular dynamics (MD) simulations along with nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) to examine how dynamics and solvent interactions contribute to the m6A recognition and negative selectivity towards unmethylated substrate. The structured water molecules surrounding the bound RNA and the methylated substrates ability to exclude bulk water molecules contribute to the YTH domains preference for m6A. Intrusions of bulk water deep into the binding pocket disrupt binding of unmethylated adenosine. The YTHDC1s preference for the 5'-Gm6A-3' motif is partially facilitated by a network of water-mediated interactions between the 2-amino group of the guanosine and residues in the m6A binding pocket. The 5'-Im6A-3' (where I is inosine) motif can be recognized too but disruption of the water network lowers affinity. The D479A mutant also disrupts the water network and destabilizes m6A binding. Our interdisciplinary study of YTHDC1 protein/RNA complex reveals an unusual physical mechanism by which solvent interactions contributes towards m6A recognition.

biophysics↗

bioRxiv2020

Insights into G-Quadruplex-Hemin Dynamics Using Atomistic Simulations: Implications for Reactivity and Folding

Guanine quadruplex nucleic acids (G4s) are involved in key biological processes such as replication or transcription. Beyond their biological relevance, G4s find applications as biotechnological tools since they readily bind hemin and enhance its peroxidase activity, creating a G4-DNAzyme. The biocatalytic properties of G4-DNAzymes have been thoroughly studied and used for biosensing purposes. Despite hundreds of applications and massive experimental efforts, the atomistic details of the reaction mechanism remain unclear. To help select between the different hypotheses currently under investigation, we use extended explicit-solvent molecular dynamics (MD) simulations to scrutinize the G4/hemin interaction. We find that besides the dominant conformation in which hemin is stacked atop the external G-quartets, hemin can also transiently bind to the loops and be brought to the external G-quartets through diverse delivery mechanisms. The simulations do not support the catalytic mechanism relying on a wobbling guanine. Similarly, catalytic role of the iron-bound water molecule is not in line with our results, however, given the simulation limitations, this observation should be considered with some caution. The simulations rather suggest tentative mechanisms in which the external G-quartet itself could be responsible for the unique H2O2-promoted biocatalytic properties of the G4/hemin complexes. Once stacked atop a terminal G-quartet, hemin rotates about its vertical axis while readily sampling shifted geometries where the iron transiently contacts oxygen atoms of the adjacent G-quartet. This dynamics is not apparent from the ensemble-averaged structure. We also visualize transient interactions between the stacked hemin and the G4 loops. Finally, we investigated interactions between hemin and on-pathway folding intermediates of the parallel-stranded G4 fold. The simulations suggest that hemin drives the folding of parallel-stranded G4s from slip-stranded intermediates, acting as a G4 chaperone. Limitations of the MD technique are briefly discussed. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/375691v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@384380org.highwire.dtl.DTLVardef@1d7c730org.highwire.dtl.DTLVardef@1562248org.highwire.dtl.DTLVardef@1e4cb9c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Phosphorothioate substitutions in RNA structure studied by molecular dynamics simulations, QM/MM calculations and NMR experiments

Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single non-bridging oxygen of the phosphate is replaced with a sulphur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as tool to explore their function. It is, however, unclear what basic physical effects PT has on RNA stability and electronic structure. Here, we present Molecular Dynamics (MD) simulations, quantum mechanical (QM) calculations, and NMR spectroscopy measurements, exploring the effects of PT modifications in the structural context of the Neomycin-sensing riboswitch (NSR). The NSR is the smallest biologically functional riboswitch with a well-defined structure stabilized by a U-turn motif. Three of the signature interactions of the U-turn; an H-bond, an anion-{pi} interaction and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA structure and dynamics. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulphur substitution, besides weakening the direct H-bond interaction, reduces the directionality of H-bonding while increasing its dispersion and induction components. It also reduces the induction and increases dispersion component of the anion-{pi} stacking. The sulphur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to unsatisfactory description of PT in simulations of the NSR. We show that it is not possible to accurately describe the PT interactions using one universal set of van der Waals sulphur parameters and provide suggestions for improving the force-field performance.

biophysics↗

bioRxiv2020

W-RESP: Well-Restrained Electrostatic Potential Derived Charges. Revisiting the Charge Derivation Model.

Representation of electrostatic interactions by a Coulombic pair-wise potential between atom-centered partial charges is a fundamental and crucial part of empirical force fields used in classical molecular dynamics simulations. The broad success of the AMBER force field family originates mainly from the restrained electrostatic potential (RESP) charge model, which derives partial charges to reproduce the electrostatic field around the molecules. However, description of the electrostatic potential around molecules by standard RESP may be biased for some types of molecules. In this study, we modified the RESP charge derivation model to improve its description of the electrostatic potential around molecules, and thus electrostatic interactions in the force field. In particular, we re-optimized the atomic radii for definition of the grid points around the molecule, redesigned the restraining scheme and included extra point charges. The RESP fitting was significantly improved for aromatic heterocyclic molecules. Thus, the suggested W-RESP(-EP) charge derivation model showed clear potential for improving the performance of the nucleic acid force fields, for which poor description of nonbonded interactions, such as underestimated base pairing, makes it difficult to describe the folding free energy landscape of small oligonucleotides.

biophysics↗

bioRxiv2020

UNCG RNA tetraloop as a formidable force-field challenge for MD simulations

Explicit solvent atomistic molecular dynamics (MD) simulations represent an established technique to study structural dynamics of RNA molecules and an important complement for diverse experimental methods. However, performance of molecular mechanical (MM) force fields (ffs) remains far from satisfactory even after decades of development, as apparent from a problematic structural description of some important RNA motifs. Actually, some of the smallest RNA molecules belong to the most challenging systems for MD simulations and, among them, the UNCG tetraloop is saliently difficult. We report a detailed analysis of UNCG MD simulations, depicting the sequence of events leading to the loss of the UNCG native state during MD simulations. We identify molecular interactions, backbone conformations and substates that are involved in the process. The total amount of MD simulation data analyzed in this work is close to 1.3 millisecond. Then, we unravel specific ff deficiencies using diverse quantum mechanical/molecular mechanical (QM/MM) and QM calculations. Comparison between the MM and QM methods shows discrepancies in the description of the 5-flanking phosphate moiety and both signature sugar-base interactions. Our work indicates that poor behavior of the UNCG tetraloop in simulations is a complex issue that cannot be attributed to one dominant and straightforwardly correctable factor. Instead, there is a concerted effect of multiple ff inaccuracies that are coupled and amplifying each other. We attempted to improve the simulation behavior by some carefully-tailored interventions but the results are still far from satisfactory, underlying the difficulties in development of accurate nucleic acids ffs.

biophysics↗

bioRxiv2020

Primitive RNA-catalysis with guanine-rich oligonucleotide sequences - the case of a (GGC)3 nonamer

A cornerstone of molecular evolution leading to the emergence of life on our planet is associated with appearance of the first catalytic RNA molecules. A question remains regarding the nature of the simplest catalytic centers that could mediate the chemistry needed for RNA-catalysis. In the current paper we provide a new example supporting our previously suggested model proposing that transiently formed open loop geometries could serve as temporary catalytic sites in the most ancient short oligonucleotides. In particular, using two independent detection techniques, PAGE and MALDI-ToF analysis, we show that prolonged thermal treatment of a 5-phosphorylated (GGC)3 sequence at weakly acidic or neutral pH in the presence of tris(hydroxymethyl)aminomethane, produces a species characterized by a (GGC)3G stoichiometry, which is compatible with the cleavage-terminal recombination chemistry suggested in our previous studies. Our new findings are complemented by microsecond-scale molecular dynamics simulations, showing that (GGC)3 dimers readily sample transient potentially catalytic geometries compatible with the experimentally observed terminal recombination chemistry.

biochemistry↗

bioRxiv2020

Fine-tuning of the AMBER RNA Force Field with a New Term Adjusting Interactions of Terminal Nucleotides

Determination of RNA structural-dynamic properties is challenging for experimental methods. Thus atomistic molecular dynamics (MD) simulations represent a helpful technique complementary to experiments. However, contemporary MD methods still suffer from limitations of force fields (ffs), including imbalances in the non-bonded ff terms. We have recently demonstrated that some improvement of state-of-the-art AMBER RNA ff can be achieved by adding a new term for H-bonding called gHBfix, which increases tuning flexibility and reduces the risk of side-effects. Still, the first gHBfix version did not fully correct simulations of short RNA tetranucleotides (TNs). TNs are key benchmark systems due to availability of unique NMR data, although giving too much weight on improving TN simulations can easily lead to over-fitting to A-form RNA. Here we combine the gHBfix version with another term called tHBfix, which separately treats H-bond interactions formed by terminal nucleotides. This allows to refine simulations of RNA TNs without affecting simulations of other RNAs. The approach is in line with adopted strategy of current RNA ffs, where the terminal nucleotides possess different parameters for the terminal atoms than the internal nucleotides. The combination of gHBfix with tHBfix significantly improves the behavior of RNA TNs during well-converged enhanced-sampling simulations. TNs mostly populate canonical A-form like states while spurious intercalated structures are largely suppressed. Still, simulations of r(AAAA) and r(UUUU) TNs show some residual discrepancies with the primary NMR data which suggests that future tuning of some other ff terms might be useful.

biophysics↗

bioRxiv2019

Stability of Two-quartet G-quadruplexes and Their Dimers in Atomistic Simulations

G-quadruplexes (GQs) are four-stranded non-canonical DNA and RNA architectures that can be formed by guanine-rich sequences. The stability of GQs increases with the number of G-quartets and three G-quartets generally form stable GQs. However, the stability of two-quartet GQs is an open issue. To understand the intrinsic stability of two-quartet GQ stems, we have carried out a series of unbiased molecular dynamics (MD) simulations ([~]505 {micro}s in total) of two- and four-quartet DNA and RNA GQs, with attention paid mainly to parallel-stranded arrangements. We used AMBER DNA parmOL15 and RNA parmOL3 force fields and tested different ion and water models. DNA two-quartet parallel-stranded GQs unfolded in all the simulations while the equivalent RNA GQ was stable in most of the simulations. GQs composed of two stacked units of two-quartet GQs were stable for both DNA and RNA. The simulations suggest that a minimum of three quartets are needed to form an intrinsically stable all-anti parallel-stranded DNA GQ. Parallel two-quartet DNA GQ may exist if substantially stabilized by another molecule or structural element, including multimerisation. On the other hand, we predict that isolated RNA two-quartet parallel GQs may form, albeit being weakly stable. We also show that ionic parameters and water models should be chosen with caution because some parameter combinations can cause spurious instability of GQ stems. Some in-so-far unnoticed limitations of force-field description of multiple ions inside the GQs are discussed, which compromise capability of simulations to fully capture the effect of increase of the number of quartets on the GQ stability.

biophysics↗

bioRxiv2019

Local-to-global signal transduction at the core of the Mn2+ sensing riboswitch

The widespread manganese-ion sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch from Xanthomonas oryzae at 2.85 [A] resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >50 s of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

biophysics↗

bioRxiv2018

IMPROVING THE PERFORMANCE OF THE RNA AMBER FORCE FIELD BY TUNING THE HYDROGEN-BONDING INTERACTIONS

Molecular dynamics (MD) simulations became a leading tool for investigation of structural dynamics of nucleic acids. Despite recent efforts to improve the empirical potentials (force fields, ffs), RNA ffs have persisting deficiencies, which hamper their utilization in quantitatively accurate simulations. Previous studies have shown that at least two salient problems contribute to difficulties in description of free-energy landscapes of small RNA motifs: (i) excessive stabilization of the unfolded single-stranded RNA ensemble by intramolecular base-phosphate and sugar-phosphate interactions, and (ii) destabilization of the native folded state by underestimation of stability of base pairing. Here, we introduce a general ff term (gHBfix) that can selectively fine-tune non-bonding interaction terms in RNA ffs, in particular the H-bonds. gHBfix potential affects the pair-wise interactions between all possible pairs of the specific atom types, while all other interactions remain intact, i.e., it is not a structure-based model. In order to probe the ability of the gHBfix potential to refine the ff non-bonded terms, we performed an extensive set of folding simulations of RNA tetranucleotides and tetraloops. Based on these data we propose particular gHBfix parameters to modify the AMBER RNA ff. The suggested parametrization significantly improves the agreement between experimental data and the simulation conformational ensembles, although our current ff version still remains far from being flawless. While attempts to tune the RNA ffs by conventional reparametrizations of dihedral potentials or non-bonded terms can lead to major undesired side effects as we demonstrate for some recently published ffs, gHBfix has a clear promising potential to improve the ff performance while avoiding introduction of major new imbalances.

biochemistry↗