Guillem Portella

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 basis of substrate-induced permeation by an amino acid antiporter

Transporters of the amino acid, polyamine and organocation (APC) superfamily play essential roles in cell redox balance, cancer, and aminoacidurias. The bacterial L-arginine/agmatine antiporter, AdiC, is the main APC structural paradigm and shares the “5 + 5 inverted repeat” fold found in other families like the Na+-coupled neurotransmitter transporters. The available AdiC crystal structures capture two states of its transport cycle: the open-to-out apo and the outward-facing Arg+-bound occluded. However, the role of Arg+ during the transition between these two states remains unknown. Here, we report the crystal structure at 3.0 Å resolution of an Arg+-bound AdiC mutant (N101A) in the open-to-out conformation, completing the picture of the major conformational states during the transport cycle of the 5 + 5 inverted repeat fold-transporters. The N101A structure is an intermediate state between the previous known AdiC conformations. The Arg+-guanidinium group in the current structure presents high mobility and delocalization, hampering substrate occlusion and resulting in a low translocation rate. Further analysis supports that proper coordination of this group with residues Asn101 and Trp293 is required to transit to the occluded state, providing the first clues on the molecular mechanism of substrate-induced fit in a 5 + 5 inverted repeat fold-transporter. The pseudosymmetry found between repeats in AdiC, and in all fold-related transporters, restraints the conformational changes, in particular the transmembrane helices rearrangements, which occur during the transport cycle. In AdiC these movements take place away from the dimer interface, explaining the independent functioning of each subunit.

Time Averaging of NMR Chemical Shifts in the MLF Peptide in the Solid State

Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density functional theory (DFT). We measured the chemical shifts of the N-formyl-L-methionyl-L-leucyl-L-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the (13)C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which accounts for the periodic nature of the crystal structure, obtaining an overall accuracy of 4.2 ppm. In order to understand the origin of the difference between experimental and calculated chemical shifts, we carried out first-principles molecular dynamics simulations to characterize the molecular motion of the MLF peptide on the picosecond time scale. We found that (13)C chemical shifts experience very rapid fluctuations of more than 20 ppm that are averaged out over less than 200 fs. Taking account of these fluctuations in the calculation of the chemical shifts resulted in an accuracy of 3.3 ppm. To investigate the effects of averaging over longer time scales we sampled the rotameric states populated by the MLF peptides in the solid state by performing a total of 5 micros classical molecular dynamics simulations. By averaging the chemical shifts over these rotameric states, we increased the accuracy of the chemical shift calculations to 3.0 ppm, with less than 1 ppm error in 10 out of 22 cases. These results suggests that better DFT-based predictions of chemical shifts of peptides and proteins will be achieved by developing improved computational strategies capable of taking into account the averaging process up to the millisecond time scale on which the chemical shift measurements report.

Structure of triplex DNA in the gas phase.

Extensive (more than 90 microseconds) molecular dynamics simulations complemented with ion-mobility mass spectrometry experiments have been used to characterize the conformational ensemble of DNA triplexes in the gas phase. Our results suggest that the ensemble of DNA triplex structures in the gas phase is well-defined over the experimental time scale, with the three strands tightly bound, and for the most abundant charge states it samples conformations only slightly more compact than the solution structure. The degree of structural alteration is however very significant, mimicking that found in duplex and much larger than that suggested for G-quadruplexes. Our data strongly supports that the gas phase triplex maintains an excellent memory of the solution structure, well-preserved helicity, and a significant number of native contacts. Once again, a linear, flexible, and charged polymer as DNA surprises us for its ability to retain three-dimensional structure in the absence of solvent. Results argue against the generally assumed roles of the different physical interactions (solvent screening of phosphate repulsion, hydrophobic effect, and solvation of accessible polar groups) in modulating the stability of DNA structures.

Chromatin Unfolding by Epigenetic Modifications Explained by Dramatic Impairment of Internucleosome Interactions: A Multiscale Computational Study

Histone tails and their epigenetic modifications play crucial roles in gene expression regulation by altering the architecture of chromatin. However, the structural mechanisms by which histone tails influence the interconversion between active and inactive chromatin remain unknown. Given the technical challenges in obtaining detailed experimental characterizations of the structure of chromatin, multiscale computations offer a promising alternative to model the effect of histone tails on chromatin folding. Here we combine multimicrosecond atomistic molecular dynamics simulations of dinucleosomes and histone tails in explicit solvent and ions, performed with three different state-of-the-art force fields and validated by experimental NMR measurements, with coarse-grained Monte Carlo simulations of 24-nucleosome arrays to describe the conformational landscape of histone tails, their roles in chromatin compaction, and the impact of lysine acetylation, a widespread epigenetic change, on both. We find that while the wild-type tails are highly flexible and disordered, the dramatic increase of secondary-structure order by lysine acetylation unfolds chromatin by decreasing tail availability for crucial fiber-compacting internucleosome interactions. This molecular level description of the effect of histone tails and their charge modifications on chromatin folding explains the sequence sensitivity and underscores the delicate connection between local and global structural and functional effects. Our approach also opens new avenues for multiscale processes of biomolecular complexes.

Conformationally rigid nucleoside probes help understand the role of sugar pucker and nucleobase orientation in the thrombin-binding aptamer.

Modified thrombin-binding aptamers carrying 2′-deoxyguanine (dG) residues with locked North- or South-bicyclo[3.1.0]hexane pseudosugars were synthesized. Individual 2′-deoxyguanosines at positions dG5, dG10, dG14 and dG15 of the aptamer were replaced by these analogues where the North/anti and South/syn conformational states were confined. It was found that the global structure of the DNA aptamer was, for the most part, very accommodating. The substitution at positions 5, 10 and 14 with a locked South/syn-dG nucleoside produced aptamers with the same stability and global structure as the innate, unmodified one. Replacing position 15 with the same South/syn-dG nucleoside induced a strong destabilization of the aptamer, while the antipodal North/anti-dG nucleoside was less destabilizing. Remarkably, the insertion of a North/anti-dG nucleoside at position 14, where both pseudosugar conformation and glycosyl torsion angle are opposite with respect to the native structure, led to the complete disruption of the G-tetraplex structure as detected by NMR and confirmed by extensive molecular dynamics simulations. We conclude that conformationally locked bicyclo[3.1.0]hexane nucleosides appear to be excellent tools for studying the role of key conformational parameters that are critical for the formation of a stable, antiparallel G-tetrad DNA structures.

MD and NMR Analyses of Choline and TMA Binding to Duplex DNA: On the Origins of Aberrant Sequence-Dependent Stability by Alkyl Cations in Aqueous and Water-Free Solvents

It has been known for decades that alkylammonium ions, such as tetramethyl ammonium (TMA), alter the usual correlation between DNA GC-content and duplex stability. In some cases it is even possible for an AT-rich duplex to be more stable than a GC-rich duplex of the same length. There has been much speculation regarding the origin of this aberration in sequence-dependent DNA duplex stability, but no clear resolution. Using a combination of molecular dynamics simulations and NMR spectroscopy we demonstrate that choline (2-hydroxy-N,N,N-trimethylethanaminium) and TMA are preferentially localized in the minor groove of DNA duplexes at A·T base pairs and these same ions show less pronounced localization in the major groove compared to what has been demonstrated for alkali and alkali earth metal ions. Furthermore, free energy calculations show that single-stranded GC-rich sequences exhibit more favorable solvation by choline than single-stranded AT-rich sequences. The sequence-specific nature of choline and TMA binding provides a rationale for the enhanced stability of AT-rich sequences when alkyl-ammonium ions are used as the counterions of DNA. Our combined theoretical and experimental study provides one of the most detailed pictures to date of cations localized along DNA in the solution state, and provides insights that go beyond understanding alkyl-ammonium ion binding to DNA. In particular, because choline and TMA bind to DNA in a manner that is found to be distinct from that previously reported for Na(+), K(+), Mg(2+), and Ca(2+), our results reveal the important but underappreciated role that most other cations play in sequence-specific duplex stability.

Understanding the connection between epigenetic DNA methylation and nucleosome positioning from computer simulations.

Cytosine methylation is one of the most important epigenetic marks that regulate the process of gene expression. Here, we have examined the effect of epigenetic DNA methylation on nucleosomal stability using molecular dynamics simulations and elastic deformation models. We found that methylation of CpG steps destabilizes nucleosomes, especially when these are placed in sites where the DNA minor groove faces the histone core. The larger stiffness of methylated CpG steps is a crucial factor behind the decrease in nucleosome stability. Methylation changes the positioning and phasing of the nucleosomal DNA, altering the accessibility of DNA to regulatory proteins, and accordingly gene functionality. Our theoretical calculations highlight a simple physical-based explanation on the foundations of epigenetic signaling.

Specific loop modifications of the thrombin‐binding aptamer trigger the formation of parallel structures

Guanine‐rich sequences show large structural variability, with folds ranging from duplex to triplex and quadruplex helices. Quadruplexes are polymorphic, and can show multiple stoichiometries, parallel and antiparallel strand alignments, and different topological arrangements. We analyze here the equilibrium between intramolecular antiparallel and intermolecular parallel G‐quadruplexes in the thrombin‐binding aptamer (TBA) sequence. Our theoretical and experimental studies demonstrate that an apparently simple modification at the loops of TBA induces a large change in the monomeric antiparallel structure of TBA to yield a parallel G‐quadruplex showing a novel T‐tetrad. The present results illustrate the extreme polymorphism of G‐quadruplexes and the ease with which their conformation in solution can be manipulated by nucleotide modification.

Structure and Dynamics of Oligonucleotides in the Gas Phase

By combining ion-mobility mass spectrometry experiments with sub-millisecond classical and ab initio molecular dynamics we fully characterized, for the first time, the dynamic ensemble of a model nucleic acid in the gas phase under electrospray ionization conditions. The studied oligonucleotide unfolds upon vaporization, loses memory of the solution structure, and explores true gas-phase conformational space. Contrary to our original expectations, the oligonucleotide shows very rich dynamics in three different timescales (multi-picosecond, nanosecond, and sub-millisecond). The shorter timescale dynamics has a quantum mechanical nature and leads to changes in the covalent structure, whereas the other two are of classical origin. Overall, this study suggests that a re-evaluation on our view of the physics of nucleic acids upon vaporization is needed.