Probing intramolecular interactions using molecular electrostatic potentials: changing electron density contours to unveil both attractive and repulsive interactions.

We focus on intramolecular interactions, using the electrostatic potential plotted on iso-density surfaces to lead the way. We show that plotting the electrostatic potential on varying iso-density envelopes much closer to the nuclei than the commonly used 0.001 a.u. contour can reveal the driving forces for such interactions, whether they be stabilizing or destabilizing. Our approach involves optimizing the structures of molecules exhibiting intramolecular interactions and then finding the contour of the electronic density which allows the interacting atoms to be separated; we call this the nearly-touching contour. The electrostatic potential allows then to identify the intramolecular interactions as either attractive or repulsive. The discussed 1,5- and 1,6-intramolecular interactions in o-bromophenol and o-nitrophenol are attractive, while the interactions between terminal methyl hydrogens in diethyl disulfides (as shown recently) and those between the closest hydrogens in planar biphenyl and phenanthrene are clearly repulsive in nature. For the attractive 1,4-interactions in trinitromethane and chlorotrinitromethane, and the 1,3-S⋯N and the 1,4-Si⋯N interactions in the ClH2Si(CH2)nNH2 series, the lack of (3,-1) bond critical points has often been cited as reason to not identify such interactions as attractive in nature. Here, by looking at the nearly-touching contours we see that bond critical points are neither necessary nor sufficient for attractive interactions, as others have pointed out, and in some instances also pointing to repulsive interactions, as the examples of planar biphenyl and phenanthrene discussed in this work show.

Intramolecular Repulsion Visible Through the Electrostatic Potential in Disulfides: Analysis via Varying Iso-density Envelopes.

For a series of diethyl disulfide conformations, the nearly touching contours of the electrostatic potential plotted on iso-density molecular surfaces allow the assessment of intramolecular repulsion. The electrostatic potential is plotted on varying iso-density envelopes to find the nearly touching contours for which (a) the surface electrostatic potential does not show overlap between atoms or functional groups and (b) the typical features are visible (σ-hole, lone pair, hydrogen VS,max). When these nearly touching contours X are closer to the nuclei, the more electron density is excluded from the iso-density envelopes and the smaller are the volumes corresponding to these envelopes. Both the contours X and the corresponding volumes are found to correlate with relative conformational energy, reflecting the degree of intramolecular repulsion present in the various diethyl disulfides. Quantitative estimates of intramolecular repulsion can be made based on relationships between the nearly touching contour X vs relative energy and volume (of the nearly touching contour X) vs relative energy, obtained for series of diethyl disulfide conformers. These relations were used to analyze intramolecular repulsion in a set of disulfides broader than diethyl disulfide conformers. We have shown that the approach of varying electronic density contours can be used in the study of repulsive intramolecular interactions, hereby extending earlier work involving attractive intermolecular interactions.

On the influence of water on THz vibrational spectral features of molecular crystals.

The nanoscale structure of molecular assemblies plays a major role in many (µ)-biological mechanisms. Molecular crystals are one of the most simple of these assemblies and are widely used in a variety of applications from pharmaceuticals and agrochemicals, to nutraceuticals and cosmetics. The collective vibrations in such molecular crystals can be probed using terahertz spectroscopy, providing unique characteristic spectral fingerprints. However, the association of the spectral features to the crystal conformation, crystal phase and its environment is a difficult task. We present a combined computational-experimental study on the incorporation of water in lactose molecular crystals, and show how simulations can be used to associate spectral features in the THz region to crystal conformations and phases. Using periodic DFT simulations of lactose molecular crystals, the role of water in the observed lactose THz spectrum is clarified, presenting both direct and indirect contributions. A specific experimental setup is built to allow the controlled heating and corresponding dehydration of the sample, providing the monitoring of the crystal phase transformation dynamics. Besides the observation that lactose phases and phase transformation appear to be more complex than previously thought - including several crystal forms in a single phase and a non-negligible water content in the so-called anhydrous phase - we draw two main conclusions from this study. Firstly, THz modes are spread over more than one molecule and require periodic computation rather than a gas-phase one. Secondly, hydration water does not only play a perturbative role but also participates in the facilitation of the THz vibrations.

Binding of Gold(III) Porphyrin by the Pro-metastatic Regulatory Protein Human Galectin-3.

Gold(III) porphyrin presents an attractive alternative to the use of, for example, cisplatin in chemotherapy. However, approaches that allow to selectively target cancer cells are highly sought. Many plant and mammalian lectins have been shown to bind oligosaccharide sequences of the aberrant glycosylation pattern found on cancerous tumors. For example human galectin-3, of the galectin family specific for ß-galactoside, is overexpressed in the extracellular matrix of tumorigenous and metastatic tissues. We searched for non-carbohydrate ligands for galectin-3 that can guide a cytotoxic drug to the cancer cells by maintaining its affinity for tumor associated carbohydrate antigens. Previous findings showed that zinc tetrasulfonatophenylporphyrin can bind galectin-3 with sub-micromolar affinity without disturbing lactose binding. Gold(III) porphyrin is not only cytotoxic to cancer cells, it knows also a potential application as photosensitiser in photodynamic therapy. We investigated the binding of gold(III) porphyrin to galectin-3 using different biophysical interaction techniques and demonstrated a low micromolar affinity of human galectin-3 for the cytotoxic compound. Co-crystallization attempts in order to understand the binding mode of gold porphyrin to galectin-3 failed, but molecular docking emphasized a highly populated secondary binding site that does not hinder lactose or Thomsen Friendenreich disaccharide binding. This suggests that gold(III) porphyrin might significantly enhance its concentration and delivery to cancer cells by binding to human galectin-3 that keeps its orientation towards tumor associated carbohydrate antigens.

Targeting Dynamical Binding Processes in the Design of Non-Antibiotic Anti-Adhesives by Molecular Simulation-The Example of FimH.

Located at the tip of type I fimbria of Escherichia coli, the bacterial adhesin FimH is responsible for the attachment of the bacteria to the (human) host by specifically binding to highly-mannosylated glycoproteins located on the exterior of the host cell wall. Adhesion represents a necessary early step in bacterial infection and specific inhibition of this process represents a valuable alternative pathway to antibiotic treatments, as such anti-adhesive drugs are non-intrusive and are therefore unlikely to induce bacterial resistance. The currently available anti-adhesives with the highest affinities for FimH still feature affinities in the nanomolar range. A prerequisite to develop higher-affinity FimH inhibitors is a molecular understanding of the FimH-inhibitor complex formation. The latest insights in the formation process are achieved by combining several molecular simulation and traditional experimental techniques. This review summarizes how molecular simulation contributed to the current knowledge of the molecular function of FimH and the importance of dynamics in the inhibitor binding process, and highlights the importance of the incorporation of dynamical aspects in (future) drug-design studies.

The Antiadhesive Strategy in Crohn's Disease: Orally Active Mannosides to Decolonize Pathogenic Escherichia coli from the Gut.

Blocking the adherence of bacteria to cells is an attractive complementary approach to current antibiotic treatments, which are faced with increasing resistance. This strategy has been particularly studied in the context of urinary tract infections (UTIs), in which the adhesion of pathogenic Escherichia coli strains to uroepithelial cells is prevented by blocking the FimH adhesin expressed at the tips of bacteria organelles called fimbriae. Recently, we extended the antiadhesive concept, showing that potent FimH antagonists can block the attachment of adherent-invasive E. coli (AIEC) colonizing the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of analogues of heptyl mannoside (HM), a previously identified nanomolar FimH inhibitor, but one that displays poor antiadhesive effects in vivo. The anomeric oxygen atom was replaced by a sulfur or a methylene group to prevent hydrolysis by intestinal glycosidases, and chemical groups were attached at the end of the alkyl tail. Importantly, a lead compound was shown to reduce AIEC levels in the feces and in the colonic and ileal mucosa after oral administration (10 mg kg(-1) ) in a transgenic mouse model of CD. The compound showed a low bioavailability, preferable in this instance, thus suggesting the possibility of setting up an innovative antiadhesive therapy, based on the water-soluble and non-cytotoxic FimH antagonists developed here, for the CD subpopulation in which AIEC plays a key role.

Convergence of atomic charges with the size of the enzymatic environment.

Atomic charges are a key concept to give more insight into the electronic structure and chemical reactivity. The Hirshfeld-I partitioning scheme applied to the model protein human 2-cysteine peroxiredoxin thioredoxin peroxidase B is used to investigate how large a protein fragment needs to be in order to achieve convergence of the atomic charge of both neutral and negatively charged residues. Convergence in atomic charges is rapidly reached for neutral residues, but not for negatively charged ones. This study pinpoints difficulties on the road toward accurate modeling of negatively charged residues of large biomolecular systems in a multiscale approach.

From thiol to sulfonic acid: modeling the oxidation pathway of protein thiols by hydrogen peroxide.

Hydrogen peroxide is a natural oxidant that can oxidize protein thiols (RSH) via sulfenic acid (RSOH) and sulfinic acid (RSO2H) to sulfonic acid (RSO3H). In this paper, we study the complete anionic and neutral oxidation pathway from thiol to sulfonic acid. Reaction barriers and reaction free energies for all three oxidation steps are computed, both for the isolated substrates and for the substrates in the presence of different model ligands (CH4, H2O, NH3) mimicking the enzymatic environment. We found for all three barriers that the anionic thiolate is more reactive than the neutral thiol. However, the assistance of the environment in the neutral pathway in a solvent-assisted proton-exchange (SAPE) mechanism can lower the reaction barrier noticeably. Polar ligands can decrease the reaction barriers, whereas apolar ligands do not influence the barrier heights. The same holds for the reaction energies: they decrease (become more negative) in the presence of polar ligands whereas apolar ligands do not have an influence. The consistently negative consecutive reaction energies for the oxidation in the anionic pathway when going from thiolate over sulfenic and sulfinic acid to sulfonic acid are in agreement with biological reversibility.

Mycoredoxin-1 is one of the missing links in the oxidative stress defence mechanism of Mycobacteria.

To survive hostile conditions, the bacterial pathogen Mycobacterium tuberculosis produces millimolar concentrations of mycothiol as a redox buffer against oxidative stress. The reductases that couple the reducing power of mycothiol to redox active proteins in the cell are not known. We report a novel mycothiol-dependent reductase (mycoredoxin-1) with a CGYC catalytic motif. With mycoredoxin-1 and mycothiol deletion strains of Mycobacterium smegmatis, we show that mycoredoxin-1 and mycothiol are involved in the protection against oxidative stress. Mycoredoxin-1 acts as an oxidoreductase exclusively linked to the mycothiol electron transfer pathway and it can reduce S-mycothiolated mixed disulphides. Moreover, we solved the solution structures of oxidized and reduced mycoredoxin-1, revealing a thioredoxin fold with a putative mycothiol-binding site. With HSQC snapshots during electron transport, we visualize the reduction of oxidized mycoredoxin-1 as a function of time and find that mycoredoxin-1 gets S-mycothiolated on its N-terminal nucleophilic cysteine. Mycoredoxin-1 has a redox potential of -218 mV and hydrogen bonding with neighbouring residues lowers the pKa of its N-terminal nucleophilic cysteine. Determination of the oxidized and reduced structures of mycoredoxin-1, better understanding of mycothiol-dependent reactions in general, will likely give new insights in how M. tuberculosis survives oxidative stress in human macrophages.

Electron capture by the thiyl radical and disulfide bond: ligand effects on the reduction potential.

The effect of non-polar and polar ligands and of monovalent cations on the one-electron reduction potential of the thiyl radical and the disulfide bond was evaluated. The reduction potentials E° for the CH3S(.)-nL/CH3S(-)-nL and CH3SSCH3-L/CH3SSCH3(.-)-L redox couples were calculated at the B3LYP, M06-2X and MP2 levels of theory, with n=1, 2 and L=CH4, C2H4, H2O, CH3OH, NH3, CH3COOH, CH3CONH2, NH4(+), Na(+), K(+) and Li(+). Non-polar ligands decrease the E° value of the thiyl radical and disulfide bond, while neutral polar ligands favour electron uptake. Charged polar ligands and cations favour electron capture by the thiyl radical while disfavouring electron uptake by the disulfide bond. Thus, the same type of ligand can have a different effect on E° depending on the redox couple. Therefore, properties of an isolated ligand cannot uniquely determine E°. The ligand effects on E° are discussed in terms of the vertical electron affinity and reorganization energy, as well as molecular orbital theory. For a given redox couple, the ligand type influences the nature of the anion formed upon electron capture and the corresponding reorganization process towards the reduced geometry.

Native glycosylation and binding of the antidepressant paroxetine in a low-resolution crystal structure of human myeloperoxidase.

Human myeloperoxidase (MPO) utilizes hydrogen peroxide to oxidize organic compounds and as such plays an essential role in cell-component synthesis, in metabolic and elimination pathways, and in the front-line defence against pathogens. Moreover, MPO is increasingly being reported to play a role in inflammation. The enzymatic activity of MPO has also been shown to depend on its glycosylation. Mammalian MPO crystal structures deposited in the Protein Data Bank (PDB) present only a partial identification of their glycosylation. Here, a newly obtained crystal structure of MPO containing four disulfide-linked dimers and showing an elaborate collection of glycans is reported. These are compared with the glycans identified in proteomics studies and from 18 human MPO structures available in the PDB. The crystal structure also contains bound paroxetine, a blocker of serotonin reuptake that has previously been identified as an irreversible inhibitor of MPO, in the presence of thiocyanate, a physiological substrate of MPO.

Histidine versus Cysteine-Bearing Heme-Dependent Halogen Peroxidases: Parallels and Differences for Cl- Oxidation.

The homodimeric myeloperoxidase (MPO) features a histidine as a proximal ligand and a sulfonium linkage covalently attaching the heme porphyrin ring to the protein. MPO is able to catalyze Cl- oxidation with about the same efficiency as chloroperoxidase at pH 7.0. In this study, we seek to explore the parallels and differences between the histidine and cysteine heme-dependent halogen peroxidases. Transition states, reaction barriers, and relevant thermodynamic properties are calculated on protein models. Together with electronic structure calculations, it gives an overview of the reaction mechanisms and of the factors that determine the selectivity between one- and two-electron paths. Conclusions point to the innate oxidizing nature of MPO with the ester and sulfonium linkages hiking up the reactivity to enable chloride oxidation. The installation of a deprotonated imidazolate as a proximal ligand does not shift the equilibrium from one- to two-electron events without influencing the chemistry of the oxidation reaction.

How thioredoxin dissociates its mixed disulfide.

The dissociation mechanism of the thioredoxin (Trx) mixed disulfide complexes is unknown and has been debated for more than twenty years. Specifically, opposing arguments for the activation of the nucleophilic cysteine as a thiolate during the dissociation of the complex have been put forward. As a key model, the complex between Trx and its endogenous substrate, arsenate reductase (ArsC), was used. In this structure, a Cys29(Trx)-Cys89(ArsC) intermediate disulfide is formed by the nucleophilic attack of Cys29(Trx) on the exposed Cys82(ArsC)-Cys89(ArsC) in oxidized ArsC. With theoretical reactivity analysis, molecular dynamics simulations, and biochemical complex formation experiments with Cys-mutants, Trx mixed disulfide dissociation was studied. We observed that the conformational changes around the intermediate disulfide bring Cys32(Trx) in contact with Cys29(Trx). Cys32(Trx) is activated for its nucleophilic attack by hydrogen bonds, and Cys32(Trx) is found to be more reactive than Cys82(ArsC). Additionally, Cys32(Trx) directs its nucleophilic attack on the more susceptible Cys29(Trx) and not on Cys89(ArsC). This multidisciplinary approach provides fresh insights into a universal thiol/disulfide exchange reaction mechanism that results in reduced substrate and oxidized Trx.

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin.

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.

Intrinsic nucleofugality scale within the framework of density functional reactivity theory.

Nucleofugality is a measure of the quality of a leaving group in substitution and elimination reactions. In a conceptual DFT context, the nucleofugality is calculated for an elaborate set of common organic leaving groups, both in the gas phase and in two organic solvents (dichloromethane and methanol). An intrinsic nucleofugality scale is constructed showing fair agreement with the classical trends in leaving group capacity in organic chemistry. The correlation of the results with acidities (tabulated pK(a) values) on one hand and experimental solvolysis reaction rate constants (kinetic parameters) on the other hand is discussed. Finally, a conceptual DFT based formula is derived, describing the influence of the solvation energy on the nucleofugality; excellent correlations were found with explicit calculations for the studied leaving groups.

How Biochemical Environments Fine-Tune a Redox Process: From Theoretical Models to Practical Applications.

In this study, we give a new physical insight into how enzymatic environments influence a redox process. This is particularly important in a biochemical context, in which oxidoreductase enzymes and low-molecular-weight cofactors create a microenvironment, fine-tuning their specific redox potential. We present a new theoretical model, quantitatively backed up by quantum chemically calculated data obtained for key biological sulfur-based model reactions involved in preserving the cellular redox homeostasis during oxidative stress. We show that environmental effects can be quantitatively predicted from the thermodynamic cycle linking ΔΔ G(OX/RED)ref-ligand values to the differential interaction energy ΔΔ Gint of the reduced and oxidized species with the environment. Our obtained data can be linked to hydrogen-bond patterns found in protein active sites. The thermodynamic model is further understood in the framework of molecular orbital theory. The key insight of this work is that the intrinsic properties of neither a redox couple nor the interacting environment (e.g., ligand) are enough by themselves to uniquely predict reduction potentials. Instead, system-environment interactions need to be considered. This study is of general interest as redox processes are pivotal to empower, protect, or damage organisms. Our presented thermodynamic model allows a pragmatically evaluation on the expected influence of a particular environment on a redox process, necessary to fully understand how redox processes take place in living organisms.

Molecular mechanism of substrate selectivity of the arginine-agmatine Antiporter AdiC.

The arginine-agmatine antiporter (AdiC) is a component of an acid resistance system developed by enteric bacteria to resist gastric acidity. In order to avoid neutral proton antiport, the monovalent form of arginine, about as abundant as its divalent form under acidic conditions, should be selectively bound by AdiC for transport into the cytosol. In this study, we shed light on the mechanism through which AdiC distinguishes Arg+ from Arg2+ of arginine by investigating the binding of both forms in addition to that of divalent agmatine, using a combination of molecular dynamics simulations with molecular and quantum mechanics calculations. We show that AdiC indeed preferentially binds Arg+. The weaker binding of divalent compounds results mostly from their greater tendency to remain hydrated than Arg+. Our data suggests that the binding of Arg+ promotes the deprotonation of Glu208, a gating residue, which in turn reinforces its interactions with AdiC, leading to longer residence times of Arg+ in the binding site. Although the total electric charge of the ligand appears to be the determinant factor in the discrimination process, two local interactions formed with Trp293, another gating residue of the binding site, also contribute to the selection mechanism: a cation-π interaction with the guanidinium group of Arg+ and an anion-π interaction involving Glu208.

How Mg2+ ions lower the SN2@P barrier in enzymatic triphosphate hydrolysis.

Our quantum chemical activation strain analyses demonstrate how Mg2+ lowers the barrier of the enzymatic triphosphate hydrolysis through two distinct mechanisms: (a) weakening of the leaving-group bond, thereby decreasing activation strain; and (b) transition state (TS) stabilization through enhanced electrophilicity of the triphosphate PPP substrate, thereby strengthening the interaction with the nucleophile.

Interplay between ion binding and catalysis in the thioredoxin-coupled arsenate reductase family.

In the thioredoxin (Trx)-coupled arsenate reductase family, arsenate reductase from Staphylococcus aureus plasmid pI258 (Sa_ArsC) and from Bacillus subtilis (Bs_ArsC) are structurally related detoxification enzymes. Catalysis of the reduction of arsenate to arsenite involves a P-loop (Cys10Thr11Gly12Asn13Ser14Cys15Arg16) structural motif and a disulphide cascade between three conserved cysteine residues (Cys10, Cys82 and Cys89). For its activity, Sa_ArsC benefits from the binding of tetrahedral oxyanions in the P-loop active site and from the binding of potassium in a specific cation-binding site. In contrast, the steady-state kinetic parameters of Bs_ArsC are not affected by sulphate or potassium. The commonly occurring mutation of a histidine (H62), located about 6 A from the potassium-binding site in Sa_ArsC, to a glutamine uncouples the kinetic dependency on potassium. In addition, the binding affinity for potassium is affected by the presence of a lysine (K33) or an aspartic acid (D33) in combination with two negative charges (D30 and E31) on the surface of Trx-coupled arsenate reductases. In the P-loop of the Trx-coupled arsenate reductase family, the peptide bond between Gly12 and Asn13 can adopt two distinct conformations. The unique geometry of the P-loop with Asn13 in beta conformation, which is not observed in structurally related LMW PTPases, is stabilized by tetrahedral oxyanions and decreases the pK(a) value of Cys10 and Cys82. Tetrahedral oxyanions stabilize the P-loop in its catalytically most active form, which might explain the observed increase in k(cat) value for Sa_ArsC. Therefore, a subtle interplay of potassium and sulphate dictates the kinetics of Trx-coupled arsenate reductases.

Mutation of Tyr137 of the universal Escherichia coli fimbrial adhesin FimH relaxes the tyrosine gate prior to mannose binding.

The most prevalent diseases manifested by Escherichia coli are acute and recurrent bladder infections and chronic inflammatory bowel diseases such as Crohn's disease. E. coli clinical isolates express the FimH adhesin, which consists of a mannose-specific lectin domain connected via a pilin domain to the tip of type 1 pili. Although the isolated FimH lectin domain has affinities in the nanomolar range for all high-mannosidic glycans, differentiation between these glycans is based on their capacity to form predominantly hydrophobic interactions within the tyrosine gate at the entrance to the binding pocket. In this study, novel crystal structures of tyrosine-gate mutants of FimH, ligand-free or in complex with heptyl α-d-O-mannopyranoside or 4-biphenyl α-d-O-mannopyranoside, are combined with quantum-mechanical calculations and molecular-dynamics simulations. In the Y48A FimH crystal structure, a large increase in the dynamics of the alkyl chain of heptyl α-d-O-mannopyranoside attempts to compensate for the absence of the aromatic ring; however, the highly energetic and stringent mannose-binding pocket of wild-type FimH is largely maintained. The Y137A mutation, on the other hand, is the most detrimental to FimH affinity and specificity: (i) in the absence of ligand the FimH C-terminal residue Thr158 intrudes into the mannose-binding pocket and (ii) ethylenediaminetetraacetic acid interacts strongly with Glu50, Thr53 and Asn136, in spite of multiple dialysis and purification steps. Upon mutation, pre-ligand-binding relaxation of the backbone dihedral angles at position 137 in the tyrosine gate and their coupling to Tyr48 via the interiorly located Ile52 form the basis of the loss of affinity of the FimH adhesin in the Y137A mutant.