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.

Molecular basis of Arginine and Lysine DNA sequence-dependent thermo-stability modulation.

We have used a variety of theoretical and experimental techniques to study the role of four basic amino acids-Arginine, Lysine, Ornithine and L-2,4-Diaminobutyric acid-on the structure, flexibility and sequence-dependent stability of DNA. We found that the presence of organic ions stabilizes the duplexes and significantly reduces the difference in stability between AT- and GC-rich duplexes with respect to the control conditions. This suggests that these amino acids, ingredients of the primordial soup during abiogenesis, could have helped to equalize the stability of AT- and GC-rich DNA oligomers, facilitating a general non-catalysed self-replication of DNA. Experiments and simulations demonstrate that organic ions have an effect that goes beyond the general electrostatic screening, involving specific interactions along the grooves of the double helix. We conclude that organic ions, largely ignored in the DNA world, should be reconsidered as crucial structural elements far from mimics of small inorganic cations.

The Impact of the HydroxyMethylCytosine epigenetic signature on DNA structure and function.

We present a comprehensive, experimental and theoretical study of the impact of 5-hydroxymethylation of DNA cytosine. Using molecular dynamics, biophysical experiments and NMR spectroscopy, we found that Ten-Eleven translocation (TET) dioxygenases generate an epigenetic variant with structural and physical properties similar to those of 5-methylcytosine. Experiments and simulations demonstrate that 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) generally lead to stiffer DNA than normal cytosine, with poorer circularization efficiencies and lower ability to form nucleosomes. In particular, we can rule out the hypothesis that hydroxymethylation reverts to unmodified cytosine physical properties, as hmC is even more rigid than mC. Thus, we do not expect dramatic changes in the chromatin structure induced by differences in physical properties between d(mCpG) and d(hmCpG). Conversely, our simulations suggest that methylated-DNA binding domains (MBDs), associated with repression activities, are sensitive to the substitution d(mCpG) ➔ d(hmCpG), while MBD3 which has a dual activation/repression activity is not sensitive to the d(mCpG) d(hmCpG) change. Overall, while gene activity changes due to cytosine methylation are the result of the combination of stiffness-related chromatin reorganization and MBD binding, those associated to 5-hydroxylation of methylcytosine could be explained by a change in the balance of repression/activation pathways related to differential MBD binding.

A multi-modal coarse grained model of DNA flexibility mappable to the atomistic level.

We present a new coarse grained method for the simulation of duplex DNA. The algorithm uses a generalized multi-harmonic model that can represent any multi-normal distribution of helical parameters, thus avoiding caveats of current mesoscopic models for DNA simulation and representing a breakthrough in the field. The method has been parameterized from accurate parmbsc1 atomistic molecular dynamics simulations of all unique tetranucleotide sequences of DNA embedded in long duplexes and takes advantage of the correlation between helical states and backbone configurations to derive atomistic representations of DNA. The algorithm, which is implemented in a simple web interface and in a standalone package reproduces with high computational efficiency the structural landscape of long segments of DNA untreatable by atomistic molecular dynamics simulations.

Modulation of the helical properties of DNA: next-to-nearest neighbour effects and beyond.

We used extensive molecular dynamics simulations to study the structural and dynamic properties of the central d(TpA) step in the highly polymorphic d(CpTpApG) tetranucleotide. Contrary to the assumption of the dinucleotide-model and its nearest neighbours (tetranucleotide-model), the properties of the central d(TpA) step change quite significantly dependent on the next-to-nearest (hexanucleotide) sequence context and in a few cases are modulated by even remote neighbours (beyond next-to-nearest from the central TpA). Our results highlight the existence of previously undescribed dynamical mechanisms for the transmission of structural information into the DNA and demonstrate the existence of certain sequences with special physical properties that can impact on the global DNA structure and dynamics.

The static and dynamic structural heterogeneities of B-DNA: extending Calladine-Dickerson rules.

We present a multi-laboratory effort to describe the structural and dynamical properties of duplex B-DNA under physiological conditions. By processing a large amount of atomistic molecular dynamics simulations, we determine the sequence-dependent structural properties of DNA as expressed in the equilibrium distribution of its stochastic dynamics. Our analysis includes a study of first and second moments of the equilibrium distribution, which can be accurately captured by a harmonic model, but with nonlocal sequence-dependence. We characterize the sequence-dependent choreography of backbone and base movements modulating the non-Gaussian or anharmonic effects manifested in the higher moments of the dynamics of the duplex when sampling the equilibrium distribution. Contrary to prior assumptions, such anharmonic deformations are not rare in DNA and can play a significant role in determining DNA conformation within complexes. Polymorphisms in helical geometries are particularly prevalent for certain tetranucleotide sequence contexts and are always coupled to a complex network of coordinated changes in the backbone. The analysis of our simulations, which contain instances of all tetranucleotide sequences, allow us to extend Calladine-Dickerson rules used for decades to interpret the average geometry of DNA, leading to a set of rules with quantitative predictive power that encompass nonlocal sequence-dependence and anharmonic fluctuations.

Molecular Determinants for Nitric Oxide Regulation of the Murine Cationic Amino Acid Transporter CAT-2A.

Cationic amino acid transporters (CATs) supply cells with essential and semiessential dibasic amino acids. Among them, l-arginine is the substrate for nitric oxide synthases (NOS) to produce nitric oxide (NO), a key signaling molecule and second messenger. In cardiac preparations, we showed that NO acutely and directly modulates transport activity by noncompetitively inhibiting these CATs. We hypothesize that this NO regulation occurs through modification of cysteine residues in CAT proteins. Homology modeling and a computational chemistry approach identified Cys347 as one of two putative targets for NO binding, of 15 Cys residues present in the low-affinity mouse CAT-2A (mCAT-2A). To test this prediction, mammalian cell lines overexpressing mCAT-2A were used for site-directed mutagenesis and uptake studies. When Cys347 was replaced with alanine (Cys347Ala), mCAT-2A became insensitive to inhibition by NO donors. In addition, the transport capacity of this variant decreased by >50% compared to that of the control, without affecting membrane expression levels or apparent affinities for the transported amino acids. Interestingly, replacing Cys347 with serine (Cys347Ser) restored uptake levels to those of the control while retaining NO insensitivity. Other Cys residues, when replaced with Ala, still produced a NO-sensitive CAT-2A. In cells co-expressing NOS and mCAT-2A, exposure to extracellular l-arginine inhibited the uptake activity of control mCAT-2A, via NO production, but not that of the Cys347Ser variant. Thus, the -SH moiety of Cys347 is largely responsible for mCAT-2A inhibition by NO. Because of the endogenous NO effect, this modulation is likely to be physiologically relevant and a potential intervention point for therapeutics.

VeriNA3d: an R package for nucleic acids data mining.

SUMMARY: veriNA3d is an R package for the analysis of nucleic acids structural data, with an emphasis in complex RNA structures. In addition to single-structure analyses, veriNA3d also implements functions to handle whole datasets of mmCIF/PDB structures that could be retrieved from public/local repositories. Our package aims to fill a gap in the data mining of nucleic acids structures to produce flexible and high throughput analysis of structural databases. AVAILABILITY AND IMPLEMENTATION: http://mmb.irbbarcelona.org/gitlab/dgallego/veriNA3d. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Allosterism and signal transfer in DNA.

We analysed the basic mechanisms of signal transmission in DNA and the origins of the allostery exhibited by systems such as the ternary complex BAMHI-DNA-GRDBD. We found that perturbation information generated by a primary protein binding event travels as a wave to distant regions of DNA following a hopping mechanism. However, such a structural perturbation is transient and does not lead to permanent changes in the DNA geometry and interaction properties at the secondary binding site. The BAMHI-DNA-GRDBD allosteric mechanism does not occur through any traditional models: direct (protein-protein), indirect (reorganization of the secondary site) readout or solvent-release. On the contrary, it is generated by a subtle and less common entropy-mediated mechanism, which might have an important role to explain other DNA-mediated cooperative effects.

Glyceraldehyde-3-phosphate dehydrogenase is a chaperone that allocates labile heme in cells.

Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.

Parmbsc1: a refined force field for DNA simulations.

We present parmbsc1, a force field for DNA atomistic simulation, which has been parameterized from high-level quantum mechanical data and tested for nearly 100 systems (representing a total simulation time of ∼ 140 µs) covering most of DNA structural space. Parmbsc1 provides high-quality results in diverse systems. Parameters and trajectories are available at http://mmb.irbbarcelona.org/ParmBSC1/.

Gene isolation and structural characterization of a legume tree defensin with a broad spectrum of antimicrobial activity.

MAIN CONCLUSION: The recombinant EcgDf1 defensin has an antimicrobial effect against both plant and human pathogens. In silico analyses predict that EcgDf1 is prone to form dimers capable of interacting with the membranes of microorganisms. Plant defensins comprise a large family of antimicrobial peptides (AMP) with a wide range of biological functions. They are cysteine-rich molecules, highly sequence diverse but with a conserved and stable structure. In this work, a defensin gene (EcgDf1) was isolated from Erythrina crista-galli, a legume tree native from South America. The predicted peptide presents eight cysteines, with a γ-core motif GXCX3-9C and six cysteines distributed like the typical defensin αß motif. The mature EcgDf1 coding sequence was heterologously expressed in Escherichia coli strains and purified by affinity chromatography. Possible dimer and oligomers of EcgDf1 were visible in SDS electrophoresis. Moreover, its 3D structure, determined by homology modeling, docking, and molecular dynamics simulations, was found to be compatible with the formation of homodimers between the ß3 and ß1-loop-α1, leaving the ß2-loop-ß3 free to interact with lipid membranes. The purified recombinant peptide inhibited the growth of several critical plant and human pathogens, like the opportunistic fungi Candida albicans and Aspergillus niger and the plant pathogens Clavibacter michiganensis ssp. michiganensis, Penicillium expansum, Botrytis cinerea, and Alternaria alternata. EcgDf1 is a promising candidate for the development of antimicrobial products for use in agriculture and medicine.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

How accurate are accurate force-fields for B-DNA?

Last generation of force-fields are raising expectations on the quality of molecular dynamics (MD) simulations of DNA, as well as to the belief that theoretical models can substitute experimental ones in several cases. However these claims are based on limited benchmarks, where MD simulations have shown the ability to reproduce already existing 'experimental models', which in turn, have an unclear accuracy to represent DNA conformation in solution. In this work we explore the ability of different force-fields to predict the structure of two new B-DNA dodecamers, determined herein by means of 1H nuclear magnetic resonance (NMR). The study allowed us to check directly for experimental NMR observables on duplexes previously not solved, and also to assess the reliability of 'experimental structures'. We observed that technical details in the annealing procedures can induce non-negligible local changes in the final structures. We also found that while not all theoretical simulations are equally reliable, those obtained using last generation of AMBER force-fields (BSC1 and BSC0OL15) show predictive power in the multi-microsecond timescale and can be safely used to reproduce global structure of DNA duplexes and fine sequence-dependent details.

Antimicrobial and structural insights of a new snakin-like peptide isolated from Peltophorum dubium (Fabaceae).

Snakins are antimicrobial peptides (AMPs) found, so far, exclusively in plants, and known to be important in the defense against a wide range of pathogens. Like other plant AMPs, they contain several positively charged amino acids, and an even number of cysteine residues forming disulfide bridges which are considered important for their usual function. Despite its importance, studies on snakin tertiary structure and mode of action are still scarce. In this study, a new snakin-like gene was isolated from the native plant Peltophorum dubium, and its expression was verified in seedlings and adult leaves. The deduced peptide (PdSN1) shows 84% sequence identity with potato snakin-1 mature peptide, with the 12 cysteines characteristic from this peptide family at the GASA domain. The mature PdSN1 coding sequence was successfully expressed in Escherichia coli. The purified recombinant peptide inhibits the growth of important plant and human pathogens, like the economically relevant potato pathogen Streptomyces scabies and the opportunistic fungi Candida albicans and Aspergillus niger. Finally, homology and ab initio modeling techniques coupled to extensive molecular dynamics simulations were used to gain insight on the 3D structure of PdSN1, which exhibited a helix-turn-helix motif conserved in both native and recombinant peptides. We found this motif to be strongly coded in the sequence of PdSN1, as it is stable under different patterns of disulfide bonds connectivity, and even when the 12 cysteines are considered in their reduced form, explaining the previous experimental evidences.

BIGNASim: a NoSQL database structure and analysis portal for nucleic acids simulation data.

Molecular dynamics simulation (MD) is, just behind genomics, the bioinformatics tool that generates the largest amounts of data, and that is using the largest amount of CPU time in supercomputing centres. MD trajectories are obtained after months of calculations, analysed in situ, and in practice forgotten. Several projects to generate stable trajectory databases have been developed for proteins, but no equivalence exists in the nucleic acids world. We present here a novel database system to store MD trajectories and analyses of nucleic acids. The initial data set available consists mainly of the benchmark of the new molecular dynamics force-field, parmBSC1. It contains 156 simulations, with over 120 µs of total simulation time. A deposition protocol is available to accept the submission of new trajectory data. The database is based on the combination of two NoSQL engines, Cassandra for storing trajectories and MongoDB to store analysis results and simulation metadata. The analyses available include backbone geometries, helical analysis, NMR observables and a variety of mechanical analyses. Individual trajectories and combined meta-trajectories can be downloaded from the portal. The system is accessible through http://mmb.irbbarcelona.org/BIGNASim/. Supplementary Material is also available on-line at http://mmb.irbbarcelona.org/BIGNASim/SuppMaterial/.

Long-timescale dynamics of the Drew-Dickerson dodecamer.

We present a systematic study of the long-timescale dynamics of the Drew-Dickerson dodecamer (DDD: d(CGCGAATTGCGC)2) a prototypical B-DNA duplex. Using our newly parameterized PARMBSC1 force field, we describe the conformational landscape of DDD in a variety of ionic environments from minimal salt to 2 M Na(+)Cl(-) or K(+)Cl(-) The sensitivity of the simulations to the use of different solvent and ion models is analyzed in detail using multi-microsecond simulations. Finally, an extended (10 µs) simulation is used to characterize slow and infrequent conformational changes in DDD, leading to the identification of previously uncharacterized conformational states of this duplex which can explain biologically relevant conformational transitions. With a total of more than 43 µs of unrestrained molecular dynamics simulation, this study is the most extensive investigation of the dynamics of the most prototypical DNA duplex.

The structural impact of DNA mismatches.

The structure and dynamics of all the transversion and transition mismatches in three different DNA environments have been characterized by molecular dynamics simulations and NMR spectroscopy. We found that the presence of mismatches produced significant local structural alterations, especially in the case of purine transversions. Mismatched pairs often show promiscuous hydrogen bonding patterns, which interchange among each other in the nanosecond time scale. This therefore defines flexible base pairs, where breathing is frequent, and where distortions in helical parameters are strong, resulting in significant alterations in groove dimension. Even if the DNA structure is plastic enough to absorb the structural impact of the mismatch, local structural changes can be propagated far from the mismatch site, following the expected through-backbone and a previously unknown through-space mechanism. The structural changes related to the presence of mismatches help to understand the different susceptibility of mismatches to the action of repairing proteins.

Direct measurement of the dielectric polarization properties of DNA.

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2-4), we found that the DNA dielectric constant is ∼ 8, considerably higher than the value of ∼ 3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson-Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

Small Details Matter: The 2'-Hydroxyl as a Conformational Switch in RNA.

While DNA is mostly a primary carrier of genetic information and displays a regular duplex structure, RNA can form very complicated and conserved 3D structures displaying a large variety of functions, such as being an intermediary carrier of the genetic information, translating such information into the protein machinery of the cell, or even acting as a chemical catalyst. At the base of such functional diversity is the subtle balance between different backbone, nucleobase, and ribose conformations, finely regulated by the combination of hydrogen bonds and stacking interactions. Although an apparently simple chemical modification, the presence of the 2'OH in RNA has a profound effect in the ribonucleotide conformational balance, adding an extra layer of complexity to the interactions network in RNA. In the present work, we have combined database analysis with extensive molecular dynamics, quantum mechanics, and hybrid QM/MM simulations to provide direct evidence on the dramatic impact of the 2'OH conformation on sugar puckering. Calculations provide evidence that proteins can modulate the 2'OH conformation to drive sugar repuckering, leading then to the formation of bioactive conformations. In summary, the 2'OH group seems to be a primary molecular switch contributing to specific protein-RNA recognition.