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Martin-Garcia F.,Molecular Modelling Group | Martin-Garcia F.,Biomol Informatics SL | Papaleo E.,Copenhagen University | Gomez-Puertas P.,Molecular Modelling Group | And 2 more authors.
PLoS ONE | Year: 2015

The continued development and utility of molecular dynamics simulations requires improvements in both the physical models used (force fields) and in our ability to sample the Boltzmann distribution of these models. Recent developments in both areas have made available multi-microsecond simulations of two proteins, ubiquitin and Protein G, using a number of different force fields. Although these force fields mostly share a common mathematical form, they differ in their parameters and in the philosophy by which these were derived, and previous analyses showed varying levels of agreement with experimental NMR data. To complement the comparison to experiments, we have performed a structural analysis of and comparison between these simulations, thereby providing insight into the relationship between force-field parameterization, the resulting ensemble of conformations and the agreement with experiments. In particular, our results show that, at a coarse level, many of the motional properties are preserved across several, though not all, force fields. At a finer level of detail, however, there are distinct differences in both the structure and dynamics of the two proteins, which can, together with comparison with experimental data, help to select force fields for simulations of proteins. A noteworthy observation is that force fields that have been reparameterized and improved to provide a more accurate energetic description of the balance between helical and coil structures are difficult to distinguish from their "unbalanced" counterparts in these simulations. This observation implies that simulations of stable, folded proteins, even those reaching 10 microseconds in length, may provide relatively little information that can be used to modify torsion parameters to achieve an accurate balance between different secondary structural elements. © 2015 Martín-García et al. Source


Oroz J.,IMDEA Madrid Institute for Advanced Studies | Valbuena A.,IMDEA Madrid Institute for Advanced Studies | Vera A.M.,IMDEA Madrid Institute for Advanced Studies | Mendieta J.,Autonomous University of Madrid | And 3 more authors.
Journal of Biological Chemistry | Year: 2011

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Reis F.P.,New University of Lisbon | Barbas A.,New University of Lisbon | Barbas A.,Institute Biologia Experimental e Tecnologica | Klauer-King A.A.,University of Texas Health Science Center at Houston | And 8 more authors.
PLoS ONE | Year: 2013

In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro , the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results. © 2013 Reis et al. Source


Marcos-Alcalde I.,Molecular Modelling Group | Setoain J.,Complutense University of Madrid | Mendieta-Moreno J.I.,Molecular Modelling Group | Mendieta-Moreno J.I.,Condensed Matter Physics Center | And 3 more authors.
Bioinformatics | Year: 2015

From conformational studies to atomistic descriptions of enzymatic reactions, potential and free energy landscapes can be used to describe biomolecular systems in detail. However, extracting the relevant data of complex 3D energy surfaces can sometimes be laborious. In this article, we present MEPSA (Minimum Energy Path Surface Analysis), a cross-platform user friendly tool for the analysis of energy landscapes from a transition state theory perspective. Some of its most relevant features are: identification of all the barriers and minima of the landscape at once, description of maxima edge profiles, detection of the lowest energy path connecting two minima and generation of transition state theory diagrams along these paths. In addition to a built-in plotting system, MEPSA can save most of the generated data into easily parseable text files, allowing more versatile uses of MEPSA's output such as the generation of molecular dynamics restraints from a calculated path. © The Author 2015. Published by Oxford University Press. All rights reserved. Source


Mendieta J.,Autonomous University of Madrid | Mendieta J.,Biomol Informatics SL | Perez-Lago L.,Autonomous University of Madrid | Perez-Lago L.,Hospital General Universitario Gregorio Maranon | And 2 more authors.
Journal of Bacteriology | Year: 2012

A bacteriophage Ø29 transcriptional regulator, protein p4, interacts with its DNA target by employing two mechanisms: by direct readout of the chemical signatures of only one DNA base and by inducing local modification on the topology of short A tracts (indirect readout). p4 binds as a dimer to targets consisting of imperfect inverted repeats. Here we used molecular dynamic simulation to define interactions of a cluster of 12 positively charged amino acids of p4 with DNA and biochemical assays with modified DNA targets and mutated proteins to quantify the contribution of residues in the nucleoprotein complex. Our results show the implication of Arg54, with non-base-specific interaction in the central A tract, in p4 binding affinity. Despite being chemically equivalent and in identical protein monomers, the two Arg54 residues differed in their interactions with DNA. We discuss an indirect-readout mechanism for p4-DNA recognition mediated by dissimilar interaction of arginines penetrating the minor groove and the inherent properties of the A tract. Our findings extend the current understanding of protein-DNA recognition and contribute to the relevance of the sequence-dependent conformational malleability of the DNA, shedding light on the role of arginines in binding affinity. Characterization of mutant p4R54A shows that the residue is required for the activity of the protein as a transcriptional regulator. © 2012, American Society for Microbiology. Source

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