Vojtěch Spiwok

Modelling of carbohydrate–aromatic interactions: ab initio energetics and force field performance

SummaryAromatic amino acid residues are often present in carbohydrate-binding sites of proteins. These binding sites are characterized by a placement of a carbohydrate moiety in a stacking orientation to an aromatic ring. This arrangement is an example of CH/π interactions. Ab initio interaction energies for 20 carbohydrate–aromatic complexes taken from 6 selected ultra-high resolution X-ray structures of glycosidases and carbohydrate-binding proteins were calculated. All interaction energies of a pyranose moiety with a side chain of an aromatic residue were calculated as attractive with interaction energy ranging from −2.8 to −12.3xa0kcal/mol as calculated at the MP2/6-311+G(d) level. Strong attractive interactions were observed for a wide range of orientations of carbohydrate and aromatic ring as present in selected X-ray structures. The most attractive interaction was associated with apparent combination of CH/π interactions and classical H-bonds. The failure of Hartree–Fock method (interaction energies from +1.0 to −6.9xa0kcal/mol) can be explained by a dispersion nature of a majority of the studied complexes. We also present a comparison of interaction energies calculated at the MP2 level with those calculated using molecular mechanics force fields (OPLS, GROMOS, CSFF/CHARMM, CHEAT/CHARMM, Glycam/AMBER, MM2 and MM3). For a majority of force fields there was a strong correlation with MP2 values. RMSD between MP2 and force field values were 1.0 for CSFF/CHARMM, 1.2 for Glycam/AMBER, 1.2 for GROMOS, 1.3 for MM3, 1.4 for MM2, 1.5 for OPLS and to 2.3 for CHEAT/CHARMM (in kcal/mol). These results show that molecular mechanics approximates interaction energies very well and support an application of molecular mechanics methods in the area of glycochemistry and glycobiology.

Metadynamics modelling of the solvent effect on primary hydroxyl rotamer equilibria in hexopyranosides.

Accurate modelling of rotamer equilibria for the primary hydroxyl groups of monosaccharides continues to be a great challenge of computational glycochemistry. The metadynamics technique was applied to study the conformational free energy surfaces of methyl alpha-D-glucopyranoside and methyl alpha-D-galactopyranoside, employing the GLYCAM06 force field. For both molecules, seven to eight conformational free-energy minima, differing in the omega (O-5-C-5-C-6-O-6) and chi (C-3-C-4-O-4-HO-4) dihedral angles, were identified in vacuum or in a water environment. The calculated rotamer equilibrium of the primary hydroxyl group is significantly different in vacuum than in water. The major effect of a water environment is the destabilisation of a hydrogen bond between O-4-HO-4 and O-6-HO-6 groups. It was possible to calculate the free-energy differences of individual rotamers with an accuracy of better than 2 kJ/mol. The calculated gg, gt and tg rotamer populations in water are in close agreement with experimental measurements, and therefore support the theoretical background of metadynamics.

Continuous metadynamics in essential coordinates as a tool for free energy modelling of conformational changes

AbstractModelling of conformational changes in biopolymers is one of the greatest challenges of molecular biophysics. Metadynamics is a recently introduced free energy modelling technique that enhances sampling of configurational (e.g. conformational) space within a molecular dynamics simulation. This enhancement is achieved by the addition of a history-dependent bias potential, which drives the system from previously visited regions. Discontinuous metadynamics in the space of essential dynamics eigenvectors (collective motions) has been proposed and tested in conformational change modelling. Here, we present an implementation of two continuous formulations of metadynamics in the essential subspace. The method was performed in a modified version of the molecular dynamics package GROMACS. These implementations were tested on conformational changes in cyclohexane, alanine dipeptide (terminally blocked alanine, Ace-Ala-Nme) and SH3 domain. The results illustrate that metadynamics in the space of essential coordinates can accurately model free energy surfaces associated with conformational changes.n FigureThe conformational free energy surface of cyclohexane in the space of the two most intensive collective motions.

Biochemical properties of three plant nucleases with anticancer potential.

Biochemical and structural properties of three recombinant (R), highly homologous, plant bifunctional nucleases from tomato (R-TBN1), hop (R-HBN1) and Arabis brassica (R-ABN1) were determined. These nucleases cleave single- and double-stranded substrates, as well as both RNA and DNA with nearly the same efficiency. In addition, they are able to cleave several artificial substrates and highly stable viroid RNA. They also possess 3-nucleotidase activity; therefore, they can be classified as nuclease I family members. Interestingly, poly(G) is resistant to cleavage and moreover it inhibits dsDNase, ssDNase and RNase activity of the studied nucleases. All three nucleases exhibit zinc-dependence and a strong stimulatory effect of Zn²+ for dsDNA cleavage. 3-D models, predicted on the basis of experimental structure of P1 nuclease, show nine amino acid residues responsible for interactions with zinc atoms, located in the same positions as in P1 nuclease. It was also shown that R-TBN1, R-HBN1, and R-ABN1 are all N-glycosylated. Oligosaccharidic chains constitute about 16% of their MW. In addition, an anticancer potential of the R-ABN1 is compared in this work with previously tested R-TBN1, and R-HBN1. R-ABN1 injected intravenously showed 70% inhibitory effect on growth of human prostate carcinoma in athymic mice.

Nonlinear vs. linear biasing in Trp-cage folding simulations

Biased simulations have great potential for the study of slow processes, including protein folding. Atomic motions in molecules are nonlinear, which suggests that simulations with enhanced sampling of collective motions traced by nonlinear dimensionality reduction methods may perform better than linear ones. In this study, we compare an unbiased folding simulation of the Trp-cage miniprotein with metadynamics simulations using both linear (principle component analysis) and nonlinear (Isomap) low dimensional embeddings as collective variables. Folding of the mini-protein was successfully simulated in 200u2009ns simulation with linear biasing and non-linear motion biasing. The folded state was correctly predicted as the free energy minimum in both simulations. We found that the advantage of linear motion biasing is that it can sample a larger conformational space, whereas the advantage of nonlinear motion biasing lies in slightly better resolution of the resulting free energy surface. In terms of sampling efficiency, both methods are comparable.

Altruistic Metadynamics: Multisystem Biased Simulation.

We present a new simple extension of multiple walker metadynamics which makes it possible to simulate simultaneously multiple different molecular systems and to predict their free energy surfaces, named Altruistic metadynamics. Similarly to basic metadynamics, it uses a bias potential in the form of hills summed over the simulation. Each system adds a big hill to its own bias potential and smaller hills to bias potentials of other systems, hence, each system enhances sampling of other systems. This makes it possible to achieve either faster reaching of the stationary point or higher accuracy of the calculated free energy surfaces. This should be efficient in modeling of series of chemically similar systems, for example, in computational drug screening by metadynamics. The method was tested on model energy surfaces, alanine dipeptide modeled in different force fields and monosaccharides of D-hexopyranose series.

Molecular simulations of hevein/(GlcNAc)3 complex with weakened OH/O and CH/π hydrogen bonds: implications for their role in complex stabilization

Carbohydrate-protein complexes are often characterized by interactions via aromatic amino acid residues. Several mechanisms have been proposed to explain these stacking-like interactions between pyranose sugars and aromatic moieties. The physical basis of these interactions is being explained as either dispersion CH/π or hydrophobic. In order to elucidate the nature of these interactions, we performed a series of molecular dynamics simulation of hevein domain (HEV32) in complex with (β-D-GlcNAc)3. Selected OH/O and CH/π hydrogen bonds involved in carbohydrate recognition were artificially weakened in 100 ns molecular dynamics simulations. Separate weakening of either OH/O or CH/π hydrogen bonds was not sufficient to destabilize the complex. This indicates that other effects, not solely CH/π dispersion interactions, contribute significantly to the stability of the complex. Significant destabilization of complexes was reached only by simultaneous weakening of OH/O and CH/π hydrogen bonds. This also shows that classical hydrogen bonds and CH/π interactions are working in concert to stabilize this carbohydrate-protein test case.

Role of Mason-Pfizer monkey virus CA-NC spacer peptide-like domain in assembly of immature particles

ABSTRACT The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroviruses. Published models of supramolecular structures of immature particles suggest that in lentiviruses and alpharetroviruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer monkey virus (M-PMV), a betaretrovirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroviruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-Å cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE Retroviruses such as HIV-1 are of great medical importance. Using Mason-Pfizer monkey virus (M-PMV) as a model retrovirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retrovirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiviruses, alpharetroviruses, and betaretroviruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroviruses, such as HIV, Rous sarcoma virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.

Global Scale Transcriptional Profiling of Two Contrasting Barley Genotypes Exposed to Moderate Drought Conditions: Contribution of Leaves and Crowns to Water Shortage Coping Strategies

Drought is a serious threat for sustainable agriculture. Barley represents a species well adapted to environmental stresses including drought. To elucidate the adaptive mechanism of barley on transcriptional level we evaluated transcriptomic changes of two contrasting barley cultivars upon drought using the microarray technique on the level of leaves and crowns. Using bioinformatic tools, differentially expressed genes in treated vs. non-treated plants were identified. Both genotypes revealed tissue dehydration under drought conditions as shown at water saturation deficit and osmotic potential data; however, dehydration was more severe in Amulet than in drought-resistant Tadmor under the same ambient conditions. Performed analysis showed that Amulet enhanced expression of genes related to active plant growth and development, while Tadmor regarding the stimulated genes revealed conservative, water saving strategy. Common reactions of both genotypes and tissues included an induction of genes encoding several stress-responsive signaling proteins, transcription factors as well as effector genes encoding proteins directly involved in stress acclimation. In leaf, tolerant cultivar effectively stimulated mainly the expression of genes encoding proteins and enzymes involved in protein folding, sulfur metabolism, ROS detoxification or lipid biosynthesis and transport. The crown specific reaction of tolerant cultivar was an enhanced expression of genes encoding proteins and enzymes involved in cell wall lignification, ABRE-dependent abscisic acid (ABA) signaling, nucleosome remodeling, along with genes for numerous jasmonate induced proteins.

Metadyn View: Fast web-based viewer of free energy surfaces calculated by metadynamics ☆

Abstract Metadynamics is a highly successful enhanced sampling technique for simulation of molecular processes and prediction of their free energy surfaces. An in-depth analysis of data obtained by this method is as important as the simulation itself. Although there are several tools to compute free energy surfaces from metadynamics data, they usually lack user friendliness and a build-in visualization part. Here we introduce Metadyn View as a fast and user friendly viewer of bias potential/free energy surfaces calculated by metadynamics in Plumed package. It is based on modern web technologies including HTML5, JavaScript and Cascade Style Sheets (CSS). It can be used by visiting the web site and uploading a HILLS file. It calculates the bias potential/free energy surface on the client-side, so it can run online or offline without necessity to install additional web engines. Moreover, it includes tools for measurement of free energies and free energy differences and data/image export. Program summary Program title: Metadyn View Catalogue identifier: AEYC_v1_0 Program summary URL: http://cpc.cs.qub.ac.uk/summaries/AEYC_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: GPL v3.0 No. of lines in distributed program, including test data, etc.: 273269 No. of bytes in distributed program, including test data, etc.: 4632839 Distribution format: tar.gz Programming language: HTML5, JavaScript, CSS, WebGL. Computer: Any computer with a modern web browser compatible with HTML5, JavaScript and CSS. Operating system: Platform-independent. RAM: Depends on the number of Gaussian hills and dimensionality of the bias potential. Classification: 3, 7.7, 23. Nature of problem: Fast and interactive visualization of free energy surfaces of molecular systems calculated by metadynamics method. Solution method: Implementation of optimized Bias Sum algorithm and a set of tools for free energy surface analysis. Unusual features: The program, due to its web-based nature, can be run on a wide range of devices and without installation. Running time: Couple of seconds for a medium sized HILLS file (tens of thousands of lines).