Identification of potential small molecule binding pockets on Rho family GTPases

Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (.100) and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.

Understanding the two-component sensing System of virulent bacteria

Report for the scientific sojourn carried out at the l’ Institute for Computational Molecular Science of the Temple University, United States, from 2010 to 2012. Two-component systems (TCS) are used by pathogenic bacteria to sense the environment within a host and activate mechanisms related to virulence and antimicrobial resistance. A prototypical example is the PhoQ/PhoP system, which is the major regulator of virulence in Salmonella. Hence, PhoQ is an attractive target for the design of new antibiotics against foodborne diseases. Inhibition of the PhoQ-mediated bacterial virulence does not result in growth inhibition, presenting less selective pressure for the generation of antibiotic resistance. Moreover, PhoQ is a histidine kinase (HK) and it is absent in animals. Nevertheless, the design of satisfactory HK inhibitors has been proven to be a challenge. To compete with the intracellular ATP concentrations, the affinity of a HK inhibidor must be in the micromolar-nanomolar range, whereas the current lead compounds have at best millimolar affinities. Moreover, the drug selectivity depends on the conformation of a highly variable loop, referred to as the “ATP-lid, which is difficult to study by X-Ray crystallography due to its flexibility. I have investigated the binding of different HK inhibitors to PhoQ. In particular, all-atom molecular dynamics simulations have been combined with enhanced sampling techniques in order to provide structural and dynamic information of the conformation of the ATP-lid. Transient interactions between these drugs and the ATP-lid have been identified and the free energy of the different binding modes has been estimated. The results obtained pinpoint the importance of protein flexibility in the HK-inhibitor binding, and constitute a first step in developing more potent and selective drugs. The computational resources of the hosting institution as well as the experience of the members of the group in drug binding and free energy methods have been crucial to carry out this work.

Distributed all-atom simulations of intrinsically unstructured proteins

The Computational Biophysics Group at the Universitat Pompeu Fabra (GRIB-UPF) hosts two unique computational resources dedicated to the execution of large scale molecular dynamics (MD) simulations: (a) the ACMD molecular-dynamics software, used on standard personal computers with graphical processing units (GPUs); and (b) the GPUGRID. net computing network, supported by users distributed worldwide that volunteer GPUs for biomedical research. We leveraged these resources and developed studies, protocols and open-source software to elucidate energetics and pathways of a number of biomolecular systems, with a special focus on flexible proteins with many degrees of freedom. First, we characterized ion permeation through the bactericidal model protein Gramicidin A conducting one of the largest studies to date with the steered MD biasing methodology. Next, we addressed an open problem in structural biology, the determination of drug-protein association kinetics; we reconstructed the binding free energy, association, and dissaciociation rates of a drug like model system through a spatial decomposition and a Makov-chain analysis. The work was published in the Proceedings of the National Academy of Sciences and become one of the few landmark papers elucidating a ligand-binding pathway. Furthermore, we investigated the unstructured Kinase Inducible Domain (KID), a 28-peptide central to signalling and transcriptional response; the kinetics of this challenging system was modelled with a Markovian approach in collaboration with Frank Noe’s group at the Freie University of Berlin. The impact of the funding includes three peer-reviewed publication on high-impact journals; three more papers under review; four MD analysis components, released as open-source software; MD protocols; didactic material, and code for the hosting group.

An assessment of gene prediction accuracy in large DNA sequences

One of the first useful products from the human genome will be a set of predicted genes. Besides its intrinsic scientific interest, the accuracy and completeness of this data set is of considerable importance for human health and medicine. Though progress has been made on computational gene identification in terms of both methods and accuracy evaluation measures, most of the sequence sets in which the programs are tested are short genomic sequences, and there is concern that these accuracy measures may not extrapolate well to larger, more challenging data sets. Given the absence of experimentally verified large genomic data sets, we constructed a semiartificial test set comprising a number of short single-gene genomic sequences with randomly generated intergenic regions. This test set, which should still present an easier problem than real human genomic sequence, mimics the approximately 200kb long BACs being sequenced. In our experiments with these longer genomic sequences, the accuracy of GENSCAN, one of the most accurate ab initio gene prediction programs, dropped significantly, although its sensitivity remained high. Conversely, the accuracy of similarity-based programs, such as GENEWISE, PROCRUSTES, and BLASTX was not affected significantly by the presence of random intergenic sequence, but depended on the strength of the similarity to the protein homolog. As expected, the accuracy dropped if the models were built using more distant homologs, and we were able to quantitatively estimate this decline. However, the specificities of these techniques are still rather good even when the similarity is weak, which is a desirable characteristic for driving expensive follow-up experiments. Our experiments suggest that though gene prediction will improve with every new protein that is discovered and through improvements in the current set of tools, we still have a long way to go before we can decipher the precise exonic structure of every gene in the human genome using purely computational methodology.

Comparative gene prediction in human and mouse

The completion of the sequencing of the mouse genome promises to help predict human genes with greater accuracy. While current ab initio gene prediction programs are remarkably sensitive (i.e., they predict at least a fragment of most genes), their specificity is often low, predicting a large number of false-positive genes in the human genome. Sequence conservation at the protein level with the mouse genome can help eliminate some of those false positives. Here we describe SGP2, a gene prediction program that combines ab initio gene prediction with TBLASTX searches between two genome sequences to provide both sensitive and specific gene predictions. The accuracy of SGP2 when used to predict genes by comparing the human and mouse genomes is assessed on a number of data sets, including single-gene data sets, the highly curated human chromosome 22 predictions, and entire genome predictions from ENSEMBL. Results indicate that SGP2 outperforms purely ab initio gene prediction methods. Results also indicate that SGP2 works about as well with 3x shotgun data as it does with fully assembled genomes. SGP2 provides a high enough specificity that its predictions can be experimentally verified at a reasonable cost. SGP2 was used to generate a complete set of gene predictions on both the human and mouse by comparing the genomes of these two species. Our results suggest that another few thousand human and mouse genes currently not in ENSEMBL are worth verifying experimentally.

T-Cell Receptors Binding Orientation over Peptide/MHC Class I Is Driven by Long-Range Interactions.

Crystallographic data about T-Cell Receptor - peptide - major histocompatibility complex class I (TCRpMHC) interaction have revealed extremely diverse TCR binding modes triggering antigen recognition. Understanding the molecular basis that governs TCR orientation over pMHC is still a considerable challenge. We present a simplified rigid approach applied on all non-redundant TCRpMHC crystal structures available. The CHARMM force field in combination with the FACTS implicit solvation model is used to study the role of long-distance interactions between the TCR and pMHC. We demonstrate that the sum of the coulomb interactions and the electrostatic solvation energies is sufficient to identify two orientations corresponding to energetic minima at 0° and 180° from the native orientation. Interestingly, these results are shown to be robust upon small structural variations of the TCR such as changes induced by Molecular Dynamics simulations, suggesting that shape complementarity is not required to obtain a reliable signal. Accurate energy minima are also identified by confronting unbound TCR crystal structures to pMHC. Furthermore, we decompose the electrostatic energy into residue contributions to estimate their role in the overall orientation. Results show that most of the driving force leading to the formation of the complex is defined by CDR1,2/MHC interactions. This long-distance contribution appears to be independent from the binding process itself, since it is reliably identified without considering neither short-range energy terms nor CDR induced fit upon binding. Ultimately, we present an attempt to predict the TCR/pMHC binding mode for a TCR structure obtained by homology modeling. The simplicity of the approach and the absence of any fitted parameters make it also easily applicable to other types of macromolecular protein complexes.

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.

Systematic study of the static electrical properties of the CO molecule: influence of the basis set size and correlation energy

The influence of the basis set size and the correlation energy in the static electrical properties of the CO molecule is assessed. In particular, we have studied both the nuclear relaxation and the vibrational contributions to the static molecular electrical properties, the vibrational Stark effect (VSE) and the vibrational intensity effect (VIE). From a mathematical point of view, when a static and uniform electric field is applied to a molecule, the energy of this system can be expressed in terms of a double power series with respect to the bond length and to the field strength. From the power series expansion of the potential energy, field-dependent expressions for the equilibrium geometry, for the potential energy and for the force constant are obtained. The nuclear relaxation and vibrational contributions to the molecular electrical properties are analyzed in terms of the derivatives of the electronic molecular properties. In general, the results presented show that accurate inclusion of the correlation energy and large basis sets are needed to calculate the molecular electrical properties and their derivatives with respect to either nuclear displacements or/and field strength. With respect to experimental data, the calculated power series coefficients are overestimated by the SCF, CISD, and QCISD methods. On the contrary, perturbation methods (MP2 and MP4) tend to underestimate them. In average and using the 6-311 + G(3df) basis set and for the CO molecule, the nuclear relaxation and the vibrational contributions to the molecular electrical properties amount to 11.7%, 3.3%, and 69.7% of the purely electronic μ, α, and β values, respectively

Enhanced EGFP Fluorescence Emission in Presence of PEG Aqueous Solutions and PIB1000-PEG6000-PIB1000 Copolymer Vesicles.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1 : 10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

Defect study of SnO2 nanostructures by cathodoluminescence analysis: Application to nanowires

Defects in SnO2 nanowires have been studied by cathodoluminescence, and the obtained spectra have been compared with those measured on SnO2 nanocrystals of different sizes in order to reveal information about point defects not determined by other characterization techniques. Dependence of the luminescence bands on the thermal treatment temperatures and pre-treatment conditions have been determined pointing out their possible relation, due to the used treatment conditions, with the oxygen vacancy concentration. To explain these cathodoluminescence spectra and their behavior, a model based on first-principles calculations of the surface oxygen vacancies in the different crystallographic directions is proposed for corroborating the existence of surface state bands localized at energy values compatible with the found cathodoluminescence bands and with the gas sensing mechanisms. CL bands centered at 1.90 and 2.20 eV are attributed to the surface oxygen vacancies 100° coordinated with tin atoms, whereas CL bands centered at 2.37 and 2.75 eV are related to the surface oxygen vacancies 130° coordinated. This combined process of cathodoluminescence and ab initio calculations is shown to be a powerful tool for nanowire defect analysis.

Measurement and simulation of anisotropy in the Infrared and Raman spectra of beta-FeSi2 single crystals

In this paper we show that the orthorhombic phase of FeSi2 (stable at room temperature) displays a sizable anisotropy in the infrared spectra, with minor effects in the Raman data too. This fact is not trivial at all, since the crystal structure corresponds to a moderate distortion of the fluorite symmetry. Our analysis is carried out on small single crystals grown by flux transport, through polarization-resolved far-infrared reflectivity and Raman measurements. Their interpretation has been obtained by means of the simulated spectra with tight-binding molecular dynamics.

Anomalous specific-heat jump in a two-component ultracold Fermi gas

The thermodynamic functions of a Fermi gas with spin population imbalance are studied in the temperature-asymmetry plane in the BCS limit. The low-temperature domain is characterized by an anomalous enhancement of the entropy and the specific heat above their values in the unpaired state, decrease of the gap and eventual unpairing phase transition as the temperature is lowered. The unpairing phase transition induces a second jump in the specific heat, which can be measured in calorimetric experiments. While the superfluid is unstable against a supercurrent carrying state, it may sustain a metastable state if cooled adiabatically down from the stable high-temperature domain. In the latter domain the temperature dependence of the gap and related functions is analogous to the predictions of the BCS theory.

Dispersionless transport in a washboard potential

We study and characterize a new dynamical regime of underdamped particles in a tilted washboard potential. We find that for small friction in a finite range of forces the particles move essentially nondispersively, that is, coherently, over long intervals of time. The associated distribution of the particle positions moves at an essentially constant velocity and is far from Gaussian-like. This new regime is complementary to, and entirely different from, well-known nonlinear response and large dispersion regimes observed for other values of the external force.

Vortices in atomic Bose-Einstein condensates in the large-gas-parameter region

In this work we compare the results of the Gross-Pitaevskii and modified Gross-Pitaevskii equations with ab initio variational Monte Carlo calculations for Bose-Einstein condensates of atoms in axially symmetric traps. We examine both the ground state and excited states having a vortex line along the z axis at high values of the gas parameter and demonstrate an excellent agreement between the modified Gross-Pitaevskii and ab initio Monte Carlo methods, both for the ground and vortex states.

Relativistic mean-field interaction with density-dependent meson-nucleon vertices based on microscopical calculations

Although ab initio calculations of relativistic Brueckner theory lead to large scalar isovector fields in nuclear matter, at present, successful versions of covariant density functional theory neglect the interactions in this channel. A new high-precision density functional DD-MEδ is presented which includes four mesons, σ, ω, δ, and ρ, with density-dependent meson-nucleon couplings. It is based to a large extent on microscopic ab initiocalculations in nuclear matter. Only four of its parameters are determined by adjusting to binding energies and charge radii of finite nuclei. The other parameters, in particular the density dependence of the meson-nucleon vertices, are adjusted to nonrelativistic and relativistic Brueckner calculations of symmetric and asymmetric nuclear matter. The isovector effective mass mp*−mn* derived from relativistic Brueckner theory is used to determine the coupling strength of the δ meson and its density dependence.