Martini 3: a general purpose force field for coarse-grained molecular dynamics.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.

Membrane mediated toppling mechanism of the folate energy coupling factor transporter.

Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients in bacteria and archaea. They consist of an integral membrane unit, the S-component, and a tripartite ECF module. It has been proposed that the S-component mediates the substrate transport by toppling over in the membrane when docking onto an ECF module. Here, we present multi-scale molecular dynamics simulations and in vitro experiments to study the molecular toppling mechanism of the S-component of a folate-specific ECF transporter. Simulations reveal a strong bending of the membrane around the ECF module that provides a driving force for toppling of the S-component. The stability of the toppled state depends on the presence of non-bilayer forming lipids, as confirmed by folate transport activity measurements. Together, our data provide evidence for a lipid-dependent toppling-based mechanism for the folate-specific ECF transporter, a mechanism that might apply to other ECF transporters.

Molecular Mechanism of Lipid Nanodisk Formation by Styrene-Maleic Acid Copolymers.

Experimental characterization of membrane proteins often requires solubilization. A recent approach is to use styrene-maleic acid (SMA) copolymers to isolate membrane proteins in nanometer-sized membrane disks, or so-called SMA lipid particles (SMALPs). The approach has the advantage of allowing direct extraction of proteins, keeping their native lipid environment. Despite the growing popularity of using SMALPs, the molecular mechanism behind the process remains poorly understood. Here, we unravel the molecular details of the nanodisk formation by using coarse-grained molecular dynamics simulations. We show how SMA copolymers bind to the lipid bilayer interface, driven by the hydrophobic effect. Due to the concerted action of multiple adsorbed copolymers, large membrane defects appear, including small, water-filled pores. The copolymers can stabilize the rim of these pores, leading to pore growth and membrane disruption. Although complete solubilization is not seen on the timescale of our simulations, self-assembly experiments show that small nanodisks are the thermodynamically preferred end state. Our findings shed light on the mechanism of SMALP formation and on their molecular structure. This can be an important step toward the design of optimized extraction tools for membrane protein research.

Targeting RNA structure in SMN2 reverses spinal muscular atrophy molecular phenotypes.

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.

Photoswitching of DNA Hybridization Using a Molecular Motor.

Reversible control over the functionality of biological systems via external triggers may be used in future medicine to reduce the need for invasive procedures. Additionally, externally regulated biomacromolecules are now considered as particularly attractive tools in nanoscience and the design of smart materials, due to their highly programmable nature and complex functionality. Incorporation of photoswitches into biomolecules, such as peptides, antibiotics, and nucleic acids, has generated exciting results in the past few years. Molecular motors offer the potential for new and more precise methods of photoregulation, due to their multistate switching cycle, unidirectionality of rotation, and helicity inversion during the rotational steps. Aided by computational studies, we designed and synthesized a photoswitchable DNA hairpin, in which a molecular motor serves as the bridgehead unit. After it was determined that motor function was not affected by the rigid arms of the linker, solid-phase synthesis was employed to incorporate the motor into an 8-base-pair self-complementary DNA strand. With the photoswitchable bridgehead in place, hairpin formation was unimpaired, while the motor part of this advanced biohybrid system retains excellent photochemical properties. Rotation of the motor generates large changes in structure, and as a consequence the duplex stability of the oligonucleotide could be regulated by UV light irradiation. Additionally, Molecular Dynamics computations were employed to rationalize the observed behavior of the motor-DNA hybrid. The results presented herein establish molecular motors as powerful multistate switches for application in biological environments.

cgHeliParm: analysis of dsDNA helical parameters for coarse-grained MARTINI molecular dynamics simulations.

SUMMARY: We introduce cgHeliParm, a python program that provides the conformational analysis of Martini-based coarse-grained double strand DNA molecules. The software calculates the helical parameters such as base, base pair and base pair step parameters. cgHeliParm can be used for the analysis of coarse grain Martini molecular dynamics trajectories without transformation into atomistic models. AVAILABILITY AND IMPLEMENTATION: This package works with Python 2.7 on MacOS and Linux. The program is freely available for download from https://github.com/ifaust83/cgheliparm. Together with the main script, the base reference files CG_X_std.lib, a number of examples and R scripts are also available from the same website. A tutorial on the use and application is also available at http://cgmartini.nl/index.php/tutorials-general-introduction/tutorial-martini-dna. CONTACT: i.faustino@rug.nl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Martini Coarse-Grained Force Field: Extension to RNA.

RNA has an important role not only as the messenger of genetic information but also as a regulator of gene expression. Given its central role in cell biology, there is significant interest in studying the structural and dynamic behavior of RNA in relation to other biomolecules. Coarse-grain molecular dynamics simulations are a key tool to that end. Here, we have extended the coarse-grain Martini force field to include RNA after our recent extension to DNA. In the same way DNA was modeled, the tertiary structure of RNA is constrained using an elastic network. This model, therefore, is not designed for applications involving RNA folding but rather offers a stable RNA structure for studying RNA interactions with other (bio)molecules. The RNA model is compatible with all other Martini models and opens the way to large-scale explicit-solvent molecular dynamics simulations of complex systems involving RNA.

Coupled binding mechanism of three sodium ions and aspartate in the glutamate transporter homologue GltTk.

Glutamate transporters catalyse the thermodynamically unfavourable transport of anionic amino acids across the cell membrane by coupling it to the downhill transport of cations. This coupling mechanism is still poorly understood, in part because the available crystal structures of these transporters are of relatively low resolution. Here we solve crystal structures of the archaeal transporter GltTk in the presence and absence of aspartate and use molecular dynamics simulations and binding assays to show how strict coupling between the binding of three sodium ions and aspartate takes place.

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/.

Molecular dynamics study of naturally existing cavity couplings in proteins.

Couplings between protein sub-structures are a common property of protein dynamics. Some of these couplings are especially interesting since they relate to function and its regulation. In this article we have studied the case of cavity couplings because cavities can host functional sites, allosteric sites, and are the locus of interactions with the cell milieu. We have divided this problem into two parts. In the first part, we have explored the presence of cavity couplings in the natural dynamics of 75 proteins, using 20 ns molecular dynamics simulations. For each of these proteins, we have obtained two trajectories around their native state. After applying a stringent filtering procedure, we found significant cavity correlations in 60% of the proteins. We analyze and discuss the structure origins of these correlations, including neighbourhood, cavity distance, etc. In the second part of our study, we have used longer simulations (≥100 ns) from the MoDEL project, to obtain a broader view of cavity couplings, particularly about their dependence on time. Using moving window computations we explored the fluctuations of cavity couplings along time, finding that these couplings could fluctuate substantially during the trajectory, reaching in several cases correlations above 0.25/0.5. In summary, we describe the structural origin and the variations with time of cavity couplings. We complete our work with a brief discussion of the biological implications of these results.

Unraveling the sequence-dependent polymorphic behavior of d(CpG) steps in B-DNA.

We have made a detailed study of one of the most surprising sources of polymorphism in B-DNA: the high twist/low twist (HT/LT) conformational change in the d(CpG) base pair step. Using extensive computations, complemented with database analysis, we were able to characterize the twist polymorphism in the d(CpG) step in all the possible tetranucleotide environment. We found that twist polymorphism is coupled with BI/BII transitions, and, quite surprisingly, with slide polymorphism in the neighboring step. Unexpectedly, the penetration of cations into the minor groove of the d(CpG) step seems to be the key element in promoting twist transitions. The tetranucleotide environment also plays an important role in the sequence-dependent d(CpG) polymorphism. In this connection, we have detected a previously unexplored intramolecular C-H···O hydrogen bond interaction that stabilizes the low twist state when 3'-purines flank the d(CpG) step. This work explains a coupled mechanism involving several apparently uncorrelated conformational transitions that has only been partially inferred by earlier experimental or theoretical studies. Our results provide a complete description of twist polymorphism in d(CpG) steps and a detailed picture of the molecular choreography associated with this conformational change.

The DNA-forming properties of 6-selenoguanine.

We present here an exhaustive characterization of the structure and properties of 6-selenoguanine, an isoster of guanine, and the impact of its introduction in DNA. This study reports the results of state-of-the-art quantum mechanical calculations and atomistic molecular dynamics simulations carried out to shed light on the impact of the replacement of guanine (G) by 6-selenoguanine (SeG) in different forms of DNA. The results point out that the G → SeG substitution leads to stable DNA duplex, antiparallel triplex and G-quadruplex structures, though local distortions are also found. These structural changes affect the thermodynamic stability of the mutation leading to a clear destabilization for all studied systems. Interestingly, the lowest effect has been found when the mutation was placed in the triplex-forming oligonucleotide strand in a reverse Hoogsteen orientation, which favours the antiparallel triplex formation regarding the G-tetraplex formation. Detailed QM studies strongly suggest that SeG impacts the HOMO-LUMO gap and accordingly the transfer properties of DNA, opening the way to modulate the conductivity properties of non-natural DNAs.

NAFlex: a web server for the study of nucleic acid flexibility.

We present NAFlex, a new web tool to study the flexibility of nucleic acids, either isolated or bound to other molecules. The server allows the user to incorporate structures from protein data banks, completing gaps and removing structural inconsistencies. It is also possible to define canonical (average or sequence-adapted) nucleic acid structures using a variety of predefined internal libraries, as well to create specific nucleic acid conformations from the sequence. The server offers a variety of methods to explore nucleic acid flexibility, such as a colorless wormlike-chain model, a base-pair resolution mesoscopic model and atomistic molecular dynamics simulations with a wide variety of protocols and force fields. The trajectories obtained by simulations, or imported externally, can be visualized and analyzed using a large number of tools, including standard Cartesian analysis, essential dynamics, helical analysis, local and global stiffness, energy decomposition, principal components and in silico NMR spectra. The server is accessible free of charge from the mmb.irbbarcelona.org/NAFlex webpage.

Functionalization of the 3'-ends of DNA and RNA strands with N-ethyl-N-coupled nucleosides: a promising approach to avoid 3'-exonuclease-catalyzed hydrolysis of therapeutic oligonucleotides.

The development of nucleic acid derivatives to generate novel medical treatments has become increasingly popular, but the high vulnerability of oligonucleotides to nucleases limits their practical use. We explored the possibility of increasing the stability against 3'-exonucleases by replacing the two 3'-terminal nucleotides by N-ethyl-N-coupled nucleosides. Molecular dynamics simulations of 3'-N-ethyl-N-modified DNA:Klenow fragment complexes suggested that this kind of alteration has negative effects on the correct positioning of the adjacent scissile phosphodiester bond at the active site of the enzyme, and accordingly was expected to protect the oligonucleotide from degradation. We verified that these modifications conferred complete resistance to 3'-exonucleases. Furthermore, cellular RNAi experiments with 3'-N-ethyl-N-modified siRNAs showed that these modifications were compatible with the RNAi machinery. Overall, our experimental and theoretical studies strongly suggest that these modified oligonucleotides could be valuable for therapeutic applications.

Improved nucleic acid descriptors for siRNA efficacy prediction.

Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of effective sequences is still a challenging task. In this article, we demonstrate that including three-dimensional descriptors improved the discrimination between active and inactive small interfering RNAs (siRNAs) in a statistical model. Five descriptor types were used: (i) nucleotide position along the siRNA sequence, (ii) nucleotide composition in terms of presence/absence of specific combinations of di- and trinucleotides, (iii) nucleotide interactions by means of a modified auto- and cross-covariance function, (iv) nucleotide thermodynamic stability derived by the nearest neighbor model representation and (v) nucleic acid structure flexibility. The duplex flexibility descriptors are derived from extended molecular dynamics simulations, which are able to describe the sequence-dependent elastic properties of RNA duplexes, even for non-standard oligonucleotides. The matrix of descriptors was analysed using three statistical packages in R (partial least squares, random forest, and support vector machine), and the most predictive model was implemented in a modeling tool we have made publicly available through SourceForge. Our implementation of new RNA descriptors coupled with appropriate statistical algorithms resulted in improved model performance for the selection of siRNA candidates when compared with publicly available siRNA prediction tools and previously published test sets. Additional validation studies based on in-house RNA interference projects confirmed the robustness of the scoring procedure in prospective studies.

Exploring polymorphisms in B-DNA helical conformations.

The traditional mesoscopic paradigm represents DNA as a series of base-pair steps whose energy response to equilibrium perturbations is elastic, with harmonic oscillations (defining local stiffness) around a single equilibrium conformation. In addition, base sequence effects are often analysed as a succession of independent XpY base-pair steps (i.e. a nearest-neighbour (NN) model with only 10 unique cases). Unfortunately, recent massive simulations carried out by the ABC consortium suggest that the real picture of DNA flexibility may be much more complex. The paradigm of DNA flexibility therefore needs to be revisited. In this article, we explore in detail one of the most obvious violations of the elastic NN model of flexibility: the bimodal distributions of some helical parameters. We perform here an in-depth statistical analysis of a very large set of MD trajectories and also of experimental structures, which lead to very solid evidence of bimodality. We then suggest ways to improve mesoscopic models to account for this deviation from the elastic regime.

Toward a consensus view of duplex RNA flexibility.

The structure and flexibility of the RNA duplex has been studied using extended molecular dynamics simulations on four diverse 18-mer oligonucleotides designed to contain many copies of the 10 unique dinucleotide steps in different sequence environments. Simulations were performed using the two most popular force fields for nucleic acids simulations (AMBER and CHARMM) in their latest versions, trying to arrive to a consensus picture of the RNA flexibility. Contrary to what was found for DNA duplex (DNA(2)), no clear convergence is found for the RNA duplex (RNA(2)), but one of the force field seems to agree better with experimental data. MD simulations performed with this force field were used to fully characterize, for the first time to our knowledge, the sequence-dependent elastic properties of RNA duplexes at different levels of resolutions. The flexibility pattern of RNA(2) shows similarities with DNA(2), but also surprising differences, which help us to understand the different biological functions of both molecules. A full mesoscopic model of RNA duplex at different resolution levels is derived to be used for genome-wide description of the flexibility of double-helical fragments of RNA.

Unique tautomeric and recognition properties of thioketothymines?

The tautomeric and recognition properties of thymine, 2- and 4-thioketothymines have been studied by means of accurate ab initio methods combined with molecular dynamics simulations and free energy calculations. In contrast to previous suggestions in the literature, the replacement of carbonyl oxygens by sulfur atoms does not lead to dramatic changes in tautomeric properties of the pyrimidine derivatives neither in vacuum nor in aqueous solution. Moreover, the presence of thioketothymines induces only mild changes in DNA structure, stability and fidelity. Despite the fact that mismatching can largely stabilize minor tautomeric forms, thioketothymines are found in the canonical thioketo-form irrespective of the paired base. Our theoretical results, confirmed by new experimental studies, describe the complete tautomeric and recognition characteristics of thioketothymines and demonstrate that both 2-thioketo and 4-thioketothymine are excellent molecules to introduce special chemical properties in modified DNA.