New Crystal Form of Human Neuropilin-1 b1 Fragment with Six Electrostatic Mutations Complexed with KDKPPR Peptide Ligand.

Neuropilin 1 (NRP1), a cell-surface co-receptor of a number of growth factors and other signaling molecules, has long been the focus of attention due to its association with the development and the progression of several types of cancer. For example, the KDKPPR peptide has recently been combined with a photosensitizer and a contrast agent to bind NRP1 for the detection and treatment by photodynamic therapy of glioblastoma, an aggressive brain cancer. The main therapeutic target is a pocket of the fragment b1 of NRP1 (NRP1-b1), in which vascular endothelial growth factors (VEGFs) bind. In the crystal packing of native human NRP1-b1, the VEGF-binding site is obstructed by a crystallographic symmetry neighbor protein, which prevents the binding of ligands. Six charged amino acids located at the protein surface were mutated to allow the protein to form a new crystal packing. The structure of the mutated fragment b1 complexed with the KDKPPR peptide was determined by X-ray crystallography. The variant crystallized in a new crystal form with the VEGF-binding cleft exposed to the solvent and, as expected, filled by the C-terminal moiety of the peptide. The atomic interactions were analyzed using new approaches based on a multipolar electron density model. Among other things, these methods indicated the role played by Asp320 and Glu348 in the electrostatic steering of the ligand in its binding site. Molecular dynamics simulations were carried out to further analyze the peptide binding and motion of the wild-type and mutant proteins. The simulations revealed that specific loops interacting with the peptide exhibited mobility in both the unbound and bound forms.

Biochemical and Structural Characterization of Chi-Class Glutathione Transferases: A Snapshot on the Glutathione Transferase Encoded by sll0067 Gene in the Cyanobacterium Synechocystis sp. Strain PCC 6803.

Glutathione transferases (GSTs) constitute a widespread superfamily of enzymes notably involved in detoxification processes and/or in specialized metabolism. In the cyanobacterium Synechocsytis sp. PCC 6803, SynGSTC1, a chi-class GST (GSTC), is thought to participate in the detoxification process of methylglyoxal, a toxic by-product of cellular metabolism. A comparative genomic analysis showed that GSTCs were present in all orders of cyanobacteria with the exception of the basal order Gloeobacterales. These enzymes were also detected in some marine and freshwater noncyanobacterial bacteria, probably as a result of horizontal gene transfer events. GSTCs were shorter of about 30 residues compared to most cytosolic GSTs and had a well-conserved SRAS motif in the active site (10SRAS13 in SynGSTC1). The crystal structure of SynGSTC1 in complex with glutathione adopted the canonical GST fold with a very open active site because the α4 and α5 helices were exceptionally short. A transferred multipolar electron-density analysis allowed a fine description of the solved structure. Unexpectedly, Ser10 did not have an electrostatic influence on glutathione as usually observed in serinyl-GSTs. The S10A variant was only slightly less efficient than the wild-type and molecular dynamics simulations suggested that S10 was a stabilizer of the protein backbone rather than an anchor site for glutathione.

Probing the Electronic Properties and Interaction Landscapes in a Series of N-(Chlorophenyl)pyridinecarboxamides.

A 3 × 3 isomer grid of nine N-(chlorophenyl)pyridinecarboxamides (NxxCl) is reported with physicochemical studies and single crystal structures (Nx = pyridinoyl moiety; xCl = aminochlorobenzene ring; x = para-/meta-/ortho-), as synthesized by the reaction of the substituted p-/m-/o-pyridinecarbonyl chlorides (Nx) with p-/m-/o-aminochlorobenzenes (xCl). Several of the nine NxxCl crystal structures display structural similarities with their halogenated NxxX and methylated NxxM relatives (x = p-/m-/o-substitutions; X = F, Br; M = methyl). Indeed, five of the nine NxxCl crystal structures are isomorphous with their NxxBr analogues as the NpmCl/Br, NpoCl/Br, NmoCl/NmoBr, NopCl/Br, and NooCl/Br pairs. In the NxxCl series, the favored hydrogen bonding mode is aggregation by N-H···Npyridine interactions, though amide···amide intermolecular interactions are noted in NpoCl and NmoCl. For the NoxCl triad, intramolecular N-H···Npyridine interactions influence molecular planarity, whereas NppCl·H2O (as a monohydrate) exhibits O-H···O, N-H···O1W, and O1W-H···N interactions as the primary hydrogen bonding. Analysis of chlorine-containing compounds on the CSD is noted for comparisons. The interaction environments are probed using Hirshfeld surface analysis and contact enrichment studies. The melting temperatures (T m) depend on both the lattice energy and molecular symmetry (Carnelley's rule), and the melting points can be well predicted from a linear regression of the two variables. The relationships of the T m values with the total energy, the electrostatic component, and the strongest hydrogen bond components have been analyzed.

A rush to explore protein-ligand electrostatic interaction energy with Charger.

The mutual penetration of electron densities between two interacting molecules complicates the computation of an accurate electrostatic interaction energy based on a pseudo-atom representation of electron densities. The numerical exact potential and multipole moment (nEP/MM) method is time-consuming since it performs a 3D integration to obtain the electrostatic energy at short interaction distances. Nguyen et al. [(2018), Acta Cryst. A74, 524-536] recently reported a fully analytical computation of the electrostatic interaction energy (aEP/MM). This method performs much faster than nEP/MM (up to two orders of magnitude) and remains highly accurate. A new program library, Charger, contains an implementation of the aEP/MM method. Charger has been incorporated into the MoProViewer software. Benchmark tests on a series of small molecules containing only C, H, N and O atoms show the efficiency of Charger in terms of execution time and accuracy. Charger is also powerful in a study of electrostatic symbiosis between a protein and a ligand. It determines reliable protein-ligand interaction energies even when both contain S atoms. It easily estimates the individual contribution of every residue to the total protein-ligand electrostatic binding energy. Glutathione transferase (GST) in complex with a benzophenone ligand was studied due to the availability of both structural and thermodynamic data. The resulting analysis highlights not only the residues that stabilize the ligand but also those that hinder ligand binding from an electrostatic point of view. This offers new perspectives in the search for mutations to improve the interaction between the two partners. A proposed mutation would improve ligand binding to GST by removing an electrostatic obstacle, rather than by the traditional increase in the number of favourable contacts.

Polarization of Electron Density Databases of Transferable Multipolar Atoms.

Polarizability is a key molecular property involved in either macroscopic (i.e., dielectric constant) and microscopic properties (i.e., interaction energies). In rigid molecules, this property only depends on the ability of the electron density (ED) to acquire electrostatic moments in response to applied electric fields. Databases of transferable electron density fragments are a cheap and efficient way to access molecular EDs. This approach is rooted in the relative conservation of the atomic ED between different molecules, termed transferability principle. The present work discusses the application of this transferability principle to the polarizability, an electron density-derived property, partitioned in atomic contributions using the Quantum Theory of Atoms In Molecules topology. The energetic consequences of accounting for in situ deformation (polarization) of database multipolar atoms are investigated in detail by using a high-quality quantum chemical benchmark.

DORI Reveals the Influence of Noncovalent Interactions on Covalent Bonding Patterns in Molecular Crystals Under Pressure.

The study of organic molecular crystals under high pressure provides fundamental insight into crystal packing distortions and reveals mechanisms of phase transitions and the crystallization of polymorphs. These solid-state transformations can be monitored directly by analyzing electron charge densities that are experimentally obtained at high pressure. However, restricting the analysis to the featureless electron density does not reveal the chemical bonding nature and the existence of intermolecular interactions. This shortcoming can be resolved by the use of the DORI (density overlap region indicator) descriptor, which is capable of simultaneously detecting both covalent patterns and noncovalent interactions from electron density and its derivatives. Using the biscarbonyl[14]annulene crystal under pressure as an example, we demonstrate how DORI can be exploited on experimental electron densities to reveal and monitor changes in electronic structure patterns resulting from molecular compression. A novel approach based on a flood-fill-type algorithm is proposed for analyzing the topology of the DORI isosurface. This approach avoids the arbitrary selection of DORI isovalues and provides an intuitive way to assess how compression packing affects covalent bonding in organic solids.

What is the relationship between the bioaccumulation of chemical contaminants in the variegated scallop Mimachlamys varia and its health status? A study carried out on the French Atlantic coast using the Path ComDim model.

Increasing activity along the French Atlantic coast has led to chronic pollution with, in particular, mixtures of contaminants such as hydrocarbons, phytosanitary products, PCBs and heavy metals. Based on previous research, pollution biomarkers were used in this study as they can indicate health status when monitoring the impact of pollutants on coastal species such as the marine bivalve Mimachlamys varia. Mollusc bivalves were sampled in March 2016, in open and semi-open areas (a harbour zone), from thirteen sites which differed in terms of their level of pollution, and were located along the Atlantic coast from Brittany down to the Nouvelle-Aquitaine region. First, analyses of heavy metals and organic contaminants (e.g. pesticides, polycyclic aromatic hydrocarbons, polychlorobiphenyl) in the digestive gland of bivalves were performed. Second, biochemical assays were used to study defence biomarkers: oxidative stress with Superoxide Dismutase (SOD), detoxification of organic compounds with Glutathione-S Transferase (GST), lipid peroxidation with Malondialdehyde (MDA), and immune processes with Laccase. In addition to the biochemical assays, a genetic approach was used to measure genetic diversity (haplotype and nucleotide diversity) at each site. Biomarker assays and genetic diversity were correlated with the chemical contaminants in bivalves using the Path-ComDim statistical model. Our results showed specific correlations between biochemical assays in the digestive glands with heavy metal contaminants, and between genetic diversity and organic pollution. Blocks of responses were analysed for correlations in order to develop standardized tools and guidelines that could improve our understanding of the short-term and long-term impact of contaminants on physiological parameters.

A method to estimate statistical errors of properties derived from charge-density modelling.

Estimating uncertainties of property values derived from a charge-density model is not straightforward. A methodology, based on calculation of sample standard deviations (SSD) of properties using randomly deviating charge-density models, is proposed with the MoPro software. The parameter shifts applied in the deviating models are generated in order to respect the variance-covariance matrix issued from the least-squares refinement. This `SSD methodology' procedure can be applied to estimate uncertainties of any property related to a charge-density model obtained by least-squares fitting. This includes topological properties such as critical point coordinates, electron density, Laplacian and ellipticity at critical points and charges integrated over atomic basins. Errors on electrostatic potentials and interaction energies are also available now through this procedure. The method is exemplified with the charge density of compound (E)-5-phenylpent-1-enylboronic acid, refined at 0.45 Šresolution. The procedure is implemented in the freely available MoPro program dedicated to charge-density refinement and modelling.

A theoretical-electron-density databank using a model of real and virtual spherical atoms.

A database describing the electron density of common chemical groups using combinations of real and virtual spherical atoms is proposed, as an alternative to the multipolar atom modelling of the molecular charge density. Theoretical structure factors were computed from periodic density functional theory calculations on 38 crystal structures of small molecules and the charge density was subsequently refined using a density model based on real spherical atoms and additional dummy charges on the covalent bonds and on electron lone-pair sites. The electron-density parameters of real and dummy atoms present in a similar chemical environment were averaged on all the molecules studied to build a database of transferable spherical atoms. Compared with the now-popular databases of transferable multipolar parameters, the spherical charge modelling needs fewer parameters to describe the molecular electron density and can be more easily incorporated in molecular modelling software for the computation of electrostatic properties. The construction method of the database is described. In order to analyse to what extent this modelling method can be used to derive meaningful molecular properties, it has been applied to the urea molecule and to biotin/streptavidin, a protein/ligand complex.

First data on three bivalve species exposed to an intra-harbour polymetallic contamination (La Rochelle, France).

Evaluating diffuse sediment contamination in the environment is a major concern with the aim of reaching a good chemical and ecological state of the littoral zone. In this study the risks of chronic chemical contamination and consequences in the bivalves Crassostrea gigas, Mytilus sp. and Mimachlamys varia were evaluated in coastal environments. The objective here was to understand the anthropological phenomena that affect the functioning of the marina of La Rochelle (semi-closed environment). Harbours seeking ecomanagement accreditations (such as the international reference ISO 14001) constitute zones of interest to implement biomonitoring studies. The biological effects of chemical pollution in the Marina of La Rochelle were studied to develop a multi-biomarker biomonitoring approach on specific marine species of this site. Moreover, a genetic (DNA barcoding) approach was applied to validate the species identity of collected bivalves. Of the three species tested the scallop, M. varia, was the most sensitive to metal exposure.

Libraries of Extremely Localized Molecular Orbitals. 1. Model Molecules Approximation and Molecular Orbitals Transferability.

Despite more and more remarkable computational ab initio results are nowadays continuously obtained for large macromolecular systems, the development of new linear-scaling techniques is still an open and stimulating field of research in theoretical chemistry. In this family of methods, an important role is occupied by those strategies based on the observation that molecules are generally constituted by recurrent functional units with well-defined intrinsic features. In this context, we propose to exploit the notion of extremely localized molecular orbitals (ELMOs) that, due to their strict localization on small molecular fragments (e.g., atoms, bonds, or functional groups), are in principle transferable from one molecule to another. Accordingly, the construction of orbital libraries to almost instantaneously build up approximate wave functions and electron densities of very large systems becomes conceivable. In this work, the ELMOs transferability is further investigated in detail and, furthermore, suitable rules to construct model molecules for the computation of ELMOs to be stored in future databanks are also defined. The obtained results confirm the reliable transferability of the ELMOs and show that electron densities obtained from the transfer of extremely localized molecular orbitals are very close to the corresponding Hartree-Fock ones. These observations prompt us to construct new ELMOs databases that could represent an alternative/complement to the already popular pseudoatoms databanks both for determining electron densities and for refining crystallographic structures of very large molecules.

Libraries of Extremely Localized Molecular Orbitals. 2. Comparison with the Pseudoatoms Transferability.

Due to both technical and methodological difficulties, determining and analyzing charge densities of very large molecular systems represents a serious challenge that, in the crystallographers community, has been mainly tackled by observing that the so-called pseudoatoms of the electron density multipole expansions are reliably transferable from molecule to molecule. This has led to the construction of pseudoatoms databanks that have allowed successful refinements of crystallographic structures of macromolecules, while taking into account their corresponding reconstructed electron distributions. A recent alternative/complement to the previous approach is represented by techniques based on extremely localized molecular orbitals (ELMOs) that, due to their strict localization on small molecular fragments (e.g., atoms, bonds, and functional groups), are also in principle exportable from system to system. The ELMOs transferability has been already tested in detail, and, in this work, it has been compared to the one of the pseudoatoms. To accomplish this task, electron distributions obtained both through the transfer of pseudoatoms and through the transfer of extremely localized molecular orbitals have been analyzed, especially taking into account topological properties and similarity indexes. The obtained results indicate that all the considered reconstruction methods give completely reasonable and similar charge densities, and, consequently, the new ELMOs libraries will probably represent new useful tools not only for refining crystal structures but also for computing approximate electronic properties of very large molecules.

Cholesterol oxidase: ultrahigh-resolution crystal structure and multipolar atom model-based analysis.

Examination of protein structure at the subatomic level is required to improve the understanding of enzymatic function. For this purpose, X-ray diffraction data have been collected at 100 K from cholesterol oxidase crystals using synchrotron radiation to an optical resolution of 0.94 Å. After refinement using the spherical atom model, nonmodelled bonding peaks were detected in the Fourier residual electron density on some of the individual bonds. Well defined bond density was observed in the peptide plane after averaging maps on the residues with the lowest thermal motion. The multipolar electron density of the protein-cofactor complex was modelled by transfer of the ELMAM2 charge-density database, and the topology of the intermolecular interactions between the protein and the flavin adenine dinucleotide (FAD) cofactor was subsequently investigated. Taking advantage of the high resolution of the structure, the stereochemistry of main-chain bond lengths and of C=O···H-N hydrogen bonds was analyzed with respect to the different secondary-structure elements.

Experimental and theoretical charge density analysis of a bromoethyl sulfonium salt.

Bromoethyl sulfonium trifluoromethanesulfonate is a salt complex in which a sulfur atom makes three covalent bonds. This molecule has been proved to act as an efficient annulation reagent which results in formation of synthetically challenging and pharmaceutically important 4-, 5-, 6-, and 7-membered heterocycles in excellent yields. The charge density of the molecule was determined from both experimentally and theoretically derived diffraction data. The stereochemistry and electron density topology of the sulfonium group was analyzed. To understand the chemical reactivity of the molecule, the electrostatic potential difference between the two carbon atoms of the bromoethyl group was investigated. It has been considered that the hydrogen atoms on the carbon atom bound to sulfur are more acidic in character due to their vicinity with the triply covalently bonded positively charged sulfur atom. The electropositivity of the S-attached and Br-attached methylene groups are compared in the experimental and theoretical charge densities using topological atomic charges and electrostatic potential at the molecular surface.

Hydrogen atoms in protein structures: high-resolution X-ray diffraction structure of the DFPase.

BACKGROUND: Hydrogen atoms represent about half of the total number of atoms in proteins and are often involved in substrate recognition and catalysis. Unfortunately, X-ray protein crystallography at usual resolution fails to access directly their positioning, mainly because light atoms display weak contributions to diffraction. However, sub-Ångstrom diffraction data, careful modeling and a proper refinement strategy can allow the positioning of a significant part of hydrogen atoms. RESULTS: A comprehensive study on the X-ray structure of the diisopropyl-fluorophosphatase (DFPase) was performed, and the hydrogen atoms were modeled, including those of solvent molecules. This model was compared to the available neutron structure of DFPase, and differences in the protein and the active site solvation were noticed. CONCLUSIONS: A further examination of the DFPase X-ray structure provides substantial evidence about the presence of an activated water molecule that may constitute an interesting piece of information as regard to the enzymatic hydrolysis mechanism.

Experimental and database-transferred electron-density analysis and evaluation of electrostatic forces in coumarin-102 dye.

The electron-density distribution of a new crystal form of coumarin-102, a laser dye, has been investigated using the Hansen-Coppens multipolar atom model. The charge density was refined versus high-resolution X-ray diffraction data collected at 100 K and was also constructed by transferring the charge density from the Experimental Library of Multipolar Atom Model (ELMAM2). The topology of the refined charge density has been analysed within the Bader `Atoms In Molecules' theory framework. Deformation electron-density peak heights and topological features indicate that the chromen-2-one ring system has a delocalized π-electron cloud in resonance with the N (amino) atom. The molecular electrostatic potential was estimated from both experimental and transferred multipolar models; it reveals an asymmetric character of the charge distribution across the molecule. This polarization effect is due to a substantial charge delocalization within the molecule. The molecular dipole moments derived from the experimental and transferred multipolar models are also compared with the liquid and gas-phase dipole moments. The substantial molecular dipole moment enhancements observed in the crystal environment originate from the crystal field and from intermolecular charge transfer induced and controlled by C-H···O and C-H···N intermolecular hydrogen bonds. The atomic forces were integrated over the atomic basins and compared for the two electron-density models.

A critical analysis of dipole-moment calculations as obtained from experimental and theoretical structure factors.

Three models of charge-density distribution - Hansen-Coppens multipolar, virtual atom and kappa - of different complexities, different numbers of refined parameters, and with variable levels of restraints, were tested against theoretical and high-resolution X-ray diffraction structure factors for 2-methyl-4-nitro-1-phenyl-1H-imidazole-5-carbonitrile. The influence of the model, refinement strategy, multipole level and treatment of the H atoms on the dipole moment was investigated. The dipole moment turned out to be very sensitive to the refinement strategy. Also, small changes in H-atom treatment can greatly influence the calculated magnitude and orientation of the dipole moment. The best results were obtained when H atoms were kept in positions determined by neutron diffraction and anisotropic displacement parameters (obtained by SHADE, in this case) were used. Also, constraints on kappa values of H atoms were found to be superior to the free refinement of these parameters. It is also shown that the over-parametrization of the multipolar model, although possibly leading to better residuals, in general gives worse dipole moments.

An improved experimental databank of transferable multipolar atom models--ELMAM2. Construction details and applications.

ELMAM2 is a generalized and improved library of experimentally derived multipolar atom types. The previously published ELMAM database is restricted mostly to protein atoms. The current database is extended to common functional groups encountered in organic molecules and is based on optimized local axes systems taking into account the local pseudosymmetry of the molecular fragment. In this approach, the symmetry-restricted multipoles have zero populations, while others take generally significant values. The various applications of the database are described. The deformation electron densities, electrostatic potentials and interaction energies calculated for several tripeptides and aromatic molecules are calculated using ELMAM2 electron-density parameters and compared with the former ELMAM database and density functional theory calculations.