Docking and homology modeling explain inhibition of the human vesicular glutamate transporters

As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three-dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three-dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol-3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.     

Cholesterol Interaction Sites on the Transmembrane Domain of the Hedgehog Signal Transducer and Class F G Protein-Coupled Receptor Smoothened

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2D-QSAR study,molecular docking,and molecular dynamics simulation studies of interaction mechanism between inhibitors and transforming growth factor-beta receptor I (ALK5)

Transforming growth factor type 1 receptor (ALK5) is kinase associated with a wide variety of pathological processes, and inhibition of ALK5 is a good strategy to treat many kinds of cancer and fibrotic diseases. Recently, a series of compounds have been synthesized as ALK5 inhibitors. However, the study of their selectivity against other potential targets remains elusive. In this research, a data-set of ALK5 inhibitors were collected and studied based on the combination of 2D-QSAR, molecular docking and molecular dynamics simulation. The quality of QSAR models were assessed statistically by F, R², and R²_ADJ, proved to be credible. The cross-validations for the models (q²_LOO = 0.571 and 0.629, respectively) showed their robustness, while the external validations (r²_test = 0.703 and 0.764, respectively) showed their predictive power. Besides, the predicted binding free energy results calculated by MM/GBSA method were in accordance with the experimental data, and the van der Waals energy term was the factor that had the most significant impact on ligand binding. What is more, several important residues were found to significantly affect the binding affinity. Finally, based on our analyses above, a proposed series of molecules were designed.     

Structure and in silico simulations of a cold-active esterase reveals its prime cold-adaptation mechanism

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7''s closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7''s cold adaptation rather than changes in dynamics.     

Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage

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Structure and mechanism of human cystine exporter cystinosin

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One membrane protein,two structures and six environments: a comparative molecular dynamics simulation study of the bacterial outer membrane protein PagP

PagP is a bacterial outer membrane protein consisting of an 8 stranded transmembrane β-barrel and an N-terminal α-helix. It is an enzyme which catalyses transfer of a palmitoyl chain from a phospholipid to lipid A. Molecular dynamics simulations have been used to compare the dynamic behaviour in simulations starting from two different structures (X-ray vs. NMR) and in six different environments (detergent micelles formed by dodecyl phosphocholine and by octyl glucoside, vs. four species of phospholipid bilayer). Analysis of interactions between the protein and its environment reveals the role played by the N-terminal α-helix, which interacts with the lipid headgroups to lock the PagP molecule into the bilayer. The PagP β-barrel adopts a tilted orientation in lipid bilayers, facilitating access of lipid tails into the mouth of the central binding pocket. In simulations starting from the X-ray structure in lipid bilayer, the L1 and L2 loops move towards one another, leading to the formation of a putative active site by residues H33, D76 and S77 coming closer together.     

Inhibitor binding studies of Mycobacterium tuberculosis MraY (Rv2156c): Insights from molecular modeling,docking, and simulation studies

Abstract

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M.tb) or tubercule bacillus, and H37Rv is the most studied clinical strain. The recent development of resistance to existing drugs is a global health-care challenge to control and cure TB. Hence, there is a critical need to discover new drug targets in M.tb. The members of peptidoglycan biosynthesis pathway are attractive target proteins for antibacterial drug development. We have performed in silico analysis of M.tb MraY (Rv2156c) integral membrane protein and constructed the three-dimensional (3D) structure model of M.tb MraY based on homology modeling method. The validated model was complexed with antibiotic muraymycin D2 (MD2) and was used to generate structure-based pharmacophore model (e-pharmacophore). High-throughput virtual screening (HTVS) of Asinex database and molecular docking of hits was performed to identify the potential inhibitors based on their mode of interactions with the key residues involved in M.tb MraY–MD2 binding. The validation of these molecules was performed using molecular dynamics (MD) simulations for two best identified hit molecules complexed with M.tb MraY in the lipid bilayer, dipalmitoylphosphatidyl-choline (DPPC) membrane. The results indicated the stability of the complexes formed and retained non-bonding interactions similar to MD2. These findings may help in the design of new inhibitors to M.tb MraY involved in peptidoglycan biosynthesis.     

Structure of the ancient TRPY1 channel from Saccharomyces cerevisiae reveals mechanisms of modulation by lipids and calcium

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Structure of 6‐diazo‐5‐oxo‐norleucine‐bound human gamma‐glutamyl transpeptidase 1, a novel mechanism of inactivation

Intense efforts are underway to identify inhibitors of the enzyme gamma‐glutamyl transpeptidase 1 (GGT1) which cleaves extracellular gamma‐glutamyl compounds and contributes to the pathology of asthma, reperfusion injury and cancer. The glutamate analog, 6‐diazo‐5‐oxo‐norleucine (DON), inhibits GGT1. DON also inhibits many essential glutamine metabolizing enzymes rendering it too toxic for use in the clinic as a GGT1 inhibitor. We investigated the molecular mechanism of human GGT1 (hGGT1) inhibition by DON to determine possible strategies for increasing its specificity for hGGT1. DON is an irreversible inhibitor of hGGT1. The second order rate constant of inactivation was 0.052 mM ^?1 min^?1 and the K _i was 2.7 ± 0.7 mM . The crystal structure of DON‐inactivated hGGT1 contained a molecule of DON without the diazo‐nitrogen atoms in the active site. The overall structure of the hGGT1‐DON complex resembled the structure of the apo‐enzyme; however, shifts were detected in the loop forming the oxyanion hole and elements of the main chain that form the entrance to the active site. The structure of hGGT1‐DON complex revealed two covalent bonds between the enzyme and inhibitor which were part of a six membered ring. The ring included the OG atom of Thr381, the reactive nucleophile of hGGT1 and the α‐amine of Thr381. The structure of DON‐bound hGGT1 has led to the discovery of a new mechanism of inactivation by DON that differs from its inactivation of other glutamine metabolizing enzymes, and insight into the activation of the catalytic nucleophile that initiates the hGGT1 reaction.     

Structure and dynamics of photosynthetic membrane-bound proteins in Rhodobacter Sphaeroides,studied with solid-state NMR spectroscopy

The photosynthetic purple bacteria such as Rb. sphaeroides possesses an intracytoplasmic membrane (ICM) and a variety of pigment-binding membrane proteins located in the ICM, acting as photoreceptor. Such photosynthetic apparatus is concentrated in the ICM. It is composed of three multimeric membrane-bound proteins; light-harvesting complexes (LH 1, LH 2), a reaction center (RC) and a cytochrome b/c1 complex. We have purified these membranes, which are called chromatophores, and characterized the structure and dynamics of the photosynthetic membrane-bound proteins by means of multi-nuclear solid state NMR. First, the isotropic chemical shift of carbonyl carbons in natural abundance and [1-¹³C] Phe labeled chromatophores indicates that the membrane-bound proteins take mainly the helical conformation. Second, the chemical shifts of side-chain resonances of uniformly ¹⁵N-labeled chromatophores indicate the side-chain histidine residue is mainly hydrogen bonded, whereas structural heterogeneity of arginine and lysine side-chains are probed by those wide distribution of ¹⁵N shifts. Thirdly, the [β-²H₃]Ala and [ε-²H₂]Tyr labeling of the chromatophores are performed and dynamics of the [β-²H]Ala and the [ε-²H₂]Tyr labeled chromatophores are studied by means of ²H solid state NMR. The dynamics of [β-²H₃]Ala is found to be a 10⁸Hz three-site jump motion with 10° liberation along the Cα-Cβ bond axis. The ²H-NMR powder pattern spectrum of [ε-²H₂] Tyr labeled chromatophores was interpreted with an averaged correlation time of 5×10⁵ Hz with 180° two-fold flips, the result of the averaging of two kinds of split spectra in terms of motional time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

Free Energy Perturbations in Ribonuclease T1 Substrate Binding. A Study of the Influence of Simulation Length,Internal Degrees of Freedom and Structure in Free Energy Perturbations

Abstract

We have studied the reliability of free energy perturbation calculations with respect to simulation protocol and simulation length in a real biological system, the binding of two different ligands to wildtype Ribonuclease T ₁ (RNT1) and to a mutant of RNT1 with Glu-46 replaced by Gln (RNT1-Gln46). The binding of the natural substrate 3′ GMP has been compared with the binding of a fluorescent probe, 2-aminopurine 3′ mono phosphate (2AP3′MP). These simulations predict that the mutant binds 2AP3′MP better than 3′GMP. Four complete free energy perturbations were performed that form a closed loop of four free energy differences, which should sum up to zero. This could be used as a tool for searching for systematic errors that are not detected by standard forward ? backward perturbations. The perturbation between 2AP3′MP and 3′GMP is quite straightforward and similar to what has been done by other groups. The perturbation between Glu46 and Gln46 is much more complex, involving as many as twelve atoms and a change of charge. This perturbation needs much longer simulation time, 500-600 ps, than used in free energy perturbations before. The increased simulation time is needed both to reach an equilibrium and to include several phases of fluctuations of the observed parameters in the production run. The extremely long simulation time is not such a severe problem as much of the work might be done on several different machines in parallel and cheap workstations are excellent for these calculations. Problems may also occur with values of the coupling parameter Λ close to 0 or 1, due to the high mobility of atoms as well as insertion/deletion in a previously unoccupied space involved in the perturbation.     

Arylpropionic acid-derived NSAIDs: New insights on derivatization,anticancer activity and potential mechanism of action

NSAIDs displayed chemopreventive and anticancer effects against several types of cancers. Moreover, combination of NSAIDs with anticancer agents resulted in enhanced anticancer activity. These findings have attracted much attention of researchers working in this field. The 2-arylpropionic acid-derived NSAIDs represent one of the most widely used anti-inflammatory agents. Additionally, they displayed antiproliferative activities against different types of cancer cells. Large volume of research was performed to identify molecular targets responsible for this activity. However, the exact mechanism underlying the anticancer activity of profens is still unclear. In this review article, the anticancer potential, structure activity relationship and synthesis of selected profen derivatives were summarized. This review is focused also on non-COX targets which can mediate the anticancer activity of this derivatives. The data in this review highlighted profens as promising lead compounds in future research to develop potent and safe anticancer agents.     

Omega-3 fatty acids and metabolic syndrome: Effects and emerging mechanisms of action

Epidemiological, human, animal, and cell culture studies show that n−3 fatty acids, especially α-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), reduce the risk factors of cardiovascular diseases. EPA and DHA, rather than ALA, have been the focus of research on the n−3 fatty acids, probably due to the relatively inefficient conversion of ALA to EPA and DHA in rodents and humans. This review will assess our current understanding of the effects and potential mechanisms of actions of individual n−3 fatty acids on multiple risk factors of metabolic syndrome. Evidence for pharmacological responses and the mechanism of action of each of the n−3 fatty acid trio will be discussed for the major risk factors of metabolic syndrome, especially adiposity, dyslipidemia, insulin resistance and diabetes, hypertension, oxidative stress, and inflammation. Metabolism of n−3 and n−6 fatty acids as well as the interactions of n−3 fatty acids with nutrients, gene expression, and disease states will be addressed to provide a rationale for the use of n−3 fatty acids to reduce the risk factors of metabolic syndrome.     

New functions of mitochondria associated membranes in cellular signaling

In all eukaryotic cells, the endoplasmic reticulum (ER) and the mitochondria establish a tight interplay, which is structurally and functionally modulated through a proteinaceous tether formed at specific subdomains of the ER membrane, designated mitochondria-associated membranes or MAMs. The tethering function of the MAMs allows the regulation of lipid synthesis and rapid transmission of calcium (Ca^2 +) signals between the ER and mitochondria, which is crucial to shape intracellular Ca^2 + signaling and regulate mitochondrial bioenergetics. Research on the molecular characterization and function of MAMs has boomed in the last few years and the list of signaling and structural proteins dynamically associated with the ER–mitochondria contact sites in physiological and pathological conditions, is rapidly increasing along with the realization of an unprecedented complexity underlying the functional role of MAMs. Besides their established role as a signaling hub for Ca^2 + and lipid transfer between ER and mitochondria, MAMs have been recently shown to regulate mitochondrial shape and motility, energy metabolism and redox status and to be central to the modulation of various key processes like ER stress, autophagy and inflammasome signaling. In this review we will discuss some emerging cell-autonomous and cell non-autonomous roles of the MAMs in mammalian cells and their relevance for important human diseases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Structural and functional properties of peptides based on the N-terminus of HIV-1 gp41 and the C-terminus of the amyloid-beta protein

Given their high alanine and glycine levels, plaque formation, α-helix to β-sheet interconversion and fusogenicity, FP (i.e., the N-terminal fusion peptide of HIV-1 gp41; 23 residues) and amyloids were proposed as belonging to the same protein superfamily. Here, we further test whether FP may exhibit ‘amyloid-like’ characteristics, by contrasting its structural and functional properties with those of Aβ(26-42), a 17-residue peptide from the C-terminus of the amyloid-beta protein responsible for Alzheimer's. FTIR spectroscopy, electron microscopy, light scattering and predicted amyloid structure aggregation (PASTA) indicated that aqueous FP and Aβ(26-42) formed similar networked β-sheet fibrils, although the FP fibril interactions were weaker. FP and Aβ(26-42) both lysed and aggregated human erythrocytes, with the hemolysis-onsets correlated with the conversion of α-helix to β-sheet for each peptide in liposomes. Congo red (CR), a marker of amyloid plaques in situ, similarly inhibited either FP- or Aβ(26-42)-induced hemolysis, and surface plasmon resonance indicated that this may be due to direct CR-peptide binding. These findings suggest that membrane-bound β-sheets of FP may contribute to the cytopathicity of HIV in vivo through an amyloid-type mechanism, and support the classification of HIV-1 FP as an ‘amyloid homolog’ (or ‘amylog’).     

Spotting the right location— imaging approaches to resolve the intracellular localization of invasive pathogens

Background

A common strategy of microbial pathogens is to invade host cells during infection. The invading microbes explore different intracellular compartments to find their preferred niche.

Scope of Review

Imaging has been instrumental to unravel paradigms of pathogen entry, to identify their exact intracellular location, and to understand the underlying mechanisms for the formation of pathogen-containing niches. Here, we provide an overview of imaging techniques that have been applied to monitor the intracellular lifestyle of pathogens, focusing mainly on bacteria that either remain in vacuolar-bound compartments or rupture the endocytic vacuole to escape into the host's cellular cytoplasm.

Major Conclusions

We will depict common molecular and cellular paradigms that are preferentially exploited by pathogens. A combination of electron microscopy, fluorescence microscopy, and time-lapse microscopy has been the driving force to reveal underlying cell biological processes. Furthermore, the development of highly sensitive and specific fluorescent sensor molecules has allowed for the identification of functional aspects of niche formation by intracellular pathogens.

General Significance

Currently, we are beginning to understand the sophistication of the invasion strategies used by bacterial pathogens during the infection process- innovative imaging has been a key ingredient for this.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.