Functional insights of two MATE transporters from Vibrio fluvialis

SummaryFunctional characterization of H- and D-MATE (Multidrug and Toxin Extrusion) transporters from clinical isolates of Vibrio fluvialis revealed H-type conferred resistance to fluoroquinolones, ethidium bromide and safranin whereas D-type exhibited marginal resistance towards ethidium bromide only. Both H-/D-type transporters were inhibited by reserpine resulting in increased intracellular norfloxacin concentration. The efflux was facilitated by both Na+/K+ ions, suggesting that these efflux pumps were ion-dependent. In presence of various classes of EPIs, there was decrease in MIC exhibited by H-/D-type efflux pumps towards norfloxacin which didn’t translate into transport inhibition. But reserpine presented a conclusive pattern with decrease in MIC towards norfloxacin and increased norfloxacin accumulation inferring maximum inhibition. Substrate binding and electrostatic charge distribution of both the transporters was similar to other known MATE transporters. The H-type exhibited 10 transmembranes and D-type exhibited 11 TMs which was different from other MATE transporters known to have 12 TMs (Transmembranes). Data derived from molecular docking and ion binding studies revealed that Aspartic Acid residue in 1st TM acts as ion binding site with transport mechanism similar to NorM. Electrostatic potential map of both the transporters revealed that there is a cavity formation within the transporters surrounded by charged electronegative amino acid residues. Interestingly, surface models of both transporters revealed that 1st TM forms covalent bond with 7th TM towards extracellular space. Docking studies also revealed that reserpine covalently binds to central pocket of both transporters and serves as excellent EPI against these transporters as evidenced by MIC and drug accumulation assays.

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A l-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily

Journal of Biological Chemistry ◽

10.1074/jbc.m113.510743 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1377-1387 ◽

Cited By ~ 8

Author(s):

Jagdeep Kaur ◽

Elena Olkhova ◽

Viveka Nand Malviya ◽

Ernst Grell ◽

Hartmut Michel

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ligand Binding ◽

Isothermal Titration Calorimetry ◽

Functional Characterization ◽

Homology Model ◽

Docking Studies ◽

Site Directed Mutagenesis ◽

Titration Calorimetry ◽

Amino Acid Residues

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of l-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of l-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of l-arginine, l-ornithine, l-2,4-diaminobutyric acid, and l-alanine. d-Lysine is bound 45 times more weakly than its l-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010–1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for l-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

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Structure of the sensory domain of McpX from Sinorhizobium meliloti, the first known bacterial chemotactic sensor for quaternary ammonium compounds

Biochemical Journal ◽

10.1042/bcj20180769 ◽

2018 ◽

Vol 475 (24) ◽

pp. 3949-3962 ◽

Cited By ~ 6

Author(s):

Manisha Shrestha ◽

Karl K. Compton ◽

Jordan M. Mancl ◽

Benjamin A. Webb ◽

Anne M. Brown ◽

...

Keyword(s):

Ligand Binding ◽

Quaternary Ammonium ◽

Sinorhizobium Meliloti ◽

Mutational Analysis ◽

Root Nodules ◽

Docking Studies ◽

Quaternary Ammonium Compounds ◽

Amino Acid Residues ◽

Binding Studies ◽

Ammonium Compounds

The α-proteobacterium Sinorhizobium meliloti can live freely in the soil or engage in a symbiosis with its legume host. S. meliloti facilitates nitrogen fixation in root nodules, thus providing pivotal, utilizable nitrogen to the host. The organism has eight chemoreceptors, namely McpT to McpZ and IcpA that facilitate chemotaxis. McpX is the first known bacterial sensor of quaternary ammonium compounds (QACs) such as choline and betaines. Because QACs are exuded at chemotaxis-relevant concentrations by germinating alfalfa seeds, McpX has been proposed to contribute to host-specific chemotaxis. We have determined the crystal structure of the McpX periplasmic region (McpXPR) in complex with the proline betaine at 2.7 Å resolution. In the crystal, the protein forms a symmetric dimer with one proline betaine molecule bound to each monomer of McpXPR within membrane-distal CACHE module. The ligand is bound through cation–πinteractions with four aromatic amino acid residues. Mutational analysis in conjunction with binding studies revealed that a conserved aspartate residue is pivotal for ligand binding. We discovered that, in a striking example of convergent evolution, the ligand-binding site of McpXPR resembles that of a group of structurally unrelated betaine-binding proteins including ProX and OpuAC. Through this comparison and docking studies, we rationalized the specificity of McpXPR for this specific group of ligands. Collectively, our structural, biochemical, and molecular docking data have revealed the molecular determinants in McpX that are crucial for its rare ligand specificity for QACs.

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Homology Modeling of Mus Musculus CDK5 and Molecular Docking Studies with Flavonoids

International Journal of Pharmaceutical and Clinical Research ◽

10.25258/ijpcr.v9i6.8779 ◽

2017 ◽

Vol 9 (6) ◽

Author(s):

Sowmya Suri ◽

Rumana Waseem ◽

Seshagiri Bandi ◽

Sania Shaik

Keyword(s):

Molecular Docking ◽

3D Model ◽

Structural Features ◽

Docking Studies ◽

Amino Acid Residues ◽

Cyclin Dependent Kinase ◽

Ligand Complex ◽

Ligand Interaction ◽

Molecular Docking Studies ◽

Cyclin Dependent Kinase 5

A 3D model of Cyclin-dependent kinase 5 (CDK5) (Accession Number: Q543f6) is generated based on crystal structure of P. falciparum PFPK5-indirubin-5-sulphonate ligand complex (PDB ID: 1V0O) at 2.30 Å resolution was used as template. Protein-ligand interaction studies were performed with flavonoids to explore structural features and binding mechanism of flavonoids as CDK5 (Cyclin-dependent kinase 5) inhibitors. The modelled structure was selected on the basis of least modeler objective function. The model was validated by PROCHECK. The predicted 3D model is reliable with 93.0% of amino acid residues in core region of the Ramachandran plot. Molecular docking studies with flavonoids viz., Diosmetin, Eriodictyol, Fortuneletin, Apigenin, Ayanin, Baicalein, Chrysoeriol and Chrysosplenol-D with modelled protein indicate that Diosmetin is the best inhibitor containing docking score of -8.23 kcal/mol. Cys83, Lys89, Asp84. The compound Diosmetin shows interactions with Cys83, Lys89, and Asp84.

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Evaluation of Cytotoxic and Tyrosinase Inhibitory Activities of 2-phenoxy(thiomethyl) pyridotriazolopyrimidines: In Vitro and Molecular Docking Studies

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200627212128 ◽

2020 ◽

Vol 20 (14) ◽

pp. 1714-1721

Author(s):

Hatem A. Abuelizz ◽

El Hassane Anouar ◽

Mohamed Marzouk ◽

Mizaton H. Hasan ◽

Siti R. Saleh ◽

...

Keyword(s):

Molecular Docking ◽

Kojic Acid ◽

Docking Studies ◽

Breast Adenocarcinoma ◽

Amino Acid Residues ◽

New Class ◽

Structure Activity ◽

Tyrosinase Inhibitors ◽

Mcf 7

Background: The use of tyrosinase has confirmed to be the best means of recognizing safe, effective, and potent tyrosinase inhibitors for whitening skin. Twenty-four 2-phenoxy(thiomethyl)pyridotriazolopyrimidines were synthesized and characterized in our previous studies. Objective: The present work aimed to evaluate their cytotoxicity against HepG2 (hepatocellular carcinoma), A549 (pulmonary adenocarcinoma), MCF-7 (breast adenocarcinoma) and WRL 68 (embryonic liver) cell lines. Methods: MTT assay was employed to investigate the cytotoxicity, and a tyrosinase inhibitor screening kit was used to evaluate the Tyrosinase (TYR) inhibitory activity of the targets. Results: The tested compounds exhibited no considerable cytotoxicity, and nine of them were selected for a tyrosinase inhibitory test. Compounds 2b, 2m, and 5a showed good inhibitory percentages against TYR compared to that of kojic acid (reference substance). Molecular docking was performed to rationalize the Structure-Activity Relationship (SAR) of the target pyridotriazolopyrimidines and analyze the binding between the docked-selected compounds and the amino acid residues in the active site of tyrosinase. Conclusion: The target pyridotriazolopyrimidines were identified as a new class of tyrosinase inhibitors.

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A Simple Method for Assessment of MDR Bacteria for Over-Expressed Efflux Pumps

The Open Microbiology Journal ◽

10.2174/1874285801307010072 ◽

2013 ◽

Vol 7 (1) ◽

pp. 72-82 ◽

Cited By ~ 39

Author(s):

Marta Martins ◽

Matthew P McCusker ◽

Miguel Viveiros ◽

Isabel Couto ◽

Séamus Fanning ◽

...

Keyword(s):

Ethidium Bromide ◽

Efflux Pump ◽

Efflux Pumps ◽

Outer Cell ◽

Gram Negative Bacteria ◽

Drug Resistant ◽

Topoisomerase Iv ◽

Simple Method ◽

Gram Negative ◽

Over Expression

It is known that bacteria showing a multi-drug resistance phenotype use several mechanisms to overcome the action of antibiotics. As a result, this phenotype can be a result of several mechanisms or a combination of thereof. The main mechanisms of antibiotic resistance are: mutations in target genes (such as DNA gyrase and topoisomerase IV); over-expression of efflux pumps; changes in the cell envelope; down regulation of membrane porins, and modified lipopolysaccharide component of the outer cell membrane (in the case of Gram-negative bacteria). In addition, adaptation to the environment, such as quorum sensing and biofilm formation can also contribute to bacterial persistence. Due to the rapid emergence and spread of bacterial isolates showing resistance to several classes of antibiotics, methods that can rapidly and efficiently identify isolates whose resistance is due to active efflux have been developed. However, there is still a need for faster and more accurate methodologies. Conventional methods that evaluate bacterial efflux pump activity in liquid systems are available. However, these methods usually use common efflux pump substrates, such as ethidium bromide or radioactive antibiotics and therefore, require specialized instrumentation, which is not available in all laboratories. In this review, we will report the results obtained with the Ethidium Bromide-agar Cartwheel method. This is an easy, instrument-free, agar based method that has been modified to afford the simultaneous evaluation of as many as twelve bacterial strains. Due to its simplicity it can be applied to large collections of bacteria to rapidly screen for multi-drug resistant isolates that show an over-expression of their efflux systems. The principle of the method is simple and relies on the ability of the bacteria to expel a fluorescent molecule that is substrate for most efflux pumps, ethidium bromide. In this approach, the higher the concentration of ethidium bromide required to produce fluorescence of the bacterial mass, the greater the efflux capacity of the bacterial cells. We have tested and applied this method to a large number of Gram-positive and Gram-negative bacteria to detect efflux activity among these multi-drug resistant isolates. The presumptive efflux activity detected by the Ethidium Bromide-agar Cartwheel method was subsequently confirmed by the determination of the minimum inhibitory concentration for several antibiotics in the presence and absence of known efflux pump inhibitors.

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Functional characterization and receptor binding studies of the malic enzyme thyroid hormone response element.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)35275-4 ◽

1991 ◽

Vol 266 (2) ◽

pp. 1008-1013 ◽

Cited By ~ 1

Author(s):

B Desvergne ◽

K J Petty ◽

V M Nikodem

Keyword(s):

Thyroid Hormone ◽

Receptor Binding ◽

Malic Enzyme ◽

Functional Characterization ◽

Hormone Response ◽

Binding Studies ◽

Response Element ◽

Hormone Response Element ◽

Thyroid Hormone Response Element

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RND Efflux Systems Contribute to Resistance and Virulence of Aliarcobacter butzleri

Antibiotics ◽

10.3390/antibiotics10070823 ◽

2021 ◽

Vol 10 (7) ◽

pp. 823

Author(s):

Cristiana Mateus ◽

Ana Rita Nunes ◽

Mónica Oleastro ◽

Fernanda Domingues ◽

Susana Ferreira

Keyword(s):

Oxidative Stress ◽

Human Serum ◽

Ethidium Bromide ◽

Biofilm Formation ◽

Bacterial Resistance ◽

Efflux Pumps ◽

Bacterial Virulence ◽

Relative Fitness ◽

Mutant Strains

Aliarcobacter butzleri is an emergent enteropathogen that can be found in a range of environments. This bacterium presents a vast repertoire of efflux pumps, such as the ones belonging to the resistance nodulation cell division family, which may be associated with bacterial resistance, as well as virulence. Thus, this work aimed to evaluate the contribution of three RND efflux systems, AreABC, AreDEF and AreGHI, in the resistance and virulence of A. butzleri. Mutant strains were constructed by inactivation of the gene that encodes the inner membrane protein of these systems. The bacterial resistance profile of parental and mutant strains to several antimicrobials was assessed, as was the intracellular accumulation of the ethidium bromide dye. Regarding bacterial virulence, the role of these three efflux pumps on growth, strain fitness, motility, biofilm formation ability, survival in adverse conditions (oxidative stress and bile salts) and human serum and in vitro adhesion and invasion to Caco-2 cells was evaluated. We observed that the mutants from the three efflux pumps were more susceptible to several classes of antimicrobials than the parental strain and presented an increase in the accumulation of ethidium bromide, indicating a potential role of the efflux pumps in the extrusion of antimicrobials. The mutant strains had no bacterial growth defects; nonetheless, they presented a reduction in relative fitness. For the three mutants, an increase in the susceptibility to oxidative stress was observed, while only the mutant for AreGHI efflux pump showed a relevant role in bile stress survival. All the mutant strains showed an impairment in biofilm formation ability, were more susceptible to human serum and were less adherent to intestinal epithelial cells. Overall, the results support the contribution of the efflux pumps AreABC, AreDEF and AreGHI of A. butzleri to antimicrobial resistance, as well as to bacterial virulence.

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A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication

Nature Communications ◽

10.1038/s41467-021-20900-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shin-ichiro Hattori ◽

Nobuyo Higashi-Kuwata ◽

Hironori Hayashi ◽

Srinivasa Rao Allu ◽

Jakka Raghavaiah ◽

...

Keyword(s):

Small Molecule ◽

Covalent Bond ◽

Amino Acid Residues ◽

X Ray ◽

Viral Breakthrough ◽

Main Protease ◽

Small Molecule Compound ◽

Polar Interactions ◽

High Concentrations

AbstractExcept remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 μM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection.

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