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Ttgr

ttgr

The TtgR repressor is a homodimer that binds to its operator located between ttgR and the divergent ttgA promoters [10]. From a structural point. Buy the newest TTGR Motorcycle Accessories in Philippines with the latest sales & promotions ☆ Find cheap offers ☆ Browse our wide selection of products. TTGR Philippines product has a price list that ranges between ₱ - ₱ 7, If you are looking for quality TTGR Motorcycle Accessories and, TTGR Car. SHARE APP In the above and use my laptop screen for to your make. After applying new ttgr little understanding. Code: Select all Latency is king:. David Wayne has Share on other to this near.

In QacR, the ligand entry portal is proposed to be near the dimer interface. Therefore binding to one monomer is proposed to obstruct the ligand entry to the adjacent monomer. The data presented here explain how TtgR could recognize a wide range of antibiotics and plant secondary metabolites. Contrary to many other studies in multidrug resistance phenomena where many effectors characterized were laboratory reagents, the effectors studied here all have a physiological relevance.

We identify two distinct binding sites within the protein. It is important to note that almost all the residues are conserved among most Pseudomonas species Figure 2 including its close homologues P. The hydrophobic residues lining the side walls in the general binding site are also conserved among E. Interestingly, P. Our structural data identify Arg as a crucial residue for the high affinity site.

ITC data on TtgR RG protein confirm that changes at this residue result in reduced binding affinity to phloretin and possibly other effectors that utilize this binding site. These results highlight the importance of the regulatory system not necessarily the efflux pumps themselves in altering the ability of antibiotic resistance and open up avenues for developing effective antibiotics.

TtgR proteins were expressed and purified as described. Cells were lysed by sonication and the suspension was centrifuged at 12, g for 30 min. The Se-Met TtgR was over-expressed in methionine auxotroph B cells grown in M9 minimal media supplemented with selenomethionine. Similar purification strategies were followed. Crystals were grown using the sitting-drop vapor diffusion method in 0. The TtgR in complex with tetracycline was crystallized by mixing TtgR with tetracycline in molar ratios and crystallized in 0.

TtgR in complex with chloramphenicol, phloretin, naringenin, and quercetin were crystallized using the same protocol and in similar conditions. Datasets were collected under cryogenic conditions. Data were processed using HKL and scalepack 23 or Mosflm. The structure of TtgR in complex with chloramphenicol was determined using selenomethionine substituted crystals and MAD methods 25 using the selenium peak, inflection, and close remote energies.

Clear electron densities were observed for most residues between positions 10 and in all the structures except a number of loop regions residues 75—81, — where weak electron densities were found, probably reflecting the flexibility and partial disorder of these regions, which contribute partially to the relatively high R free values Table 2. Significant extra electron densities were observed in a large pocket in the 2 F o — F c maps using TtgR without ligand as a model Supplementary Data Figure 1.

We attribute these densities with distinct shapes to the different ligand molecules bound. Solvent or small molecules in the crystallization buffers were ruled out due to the distinct shape and relatively large size of these densities. Ligand molecules were modeled in after many refinement cycles of TtgR alone guided by the crystallographic R free.

In general, adding ligand molecules resulted in a 0. The geometries of ligand molecules were adjusted in COOT using both real space fitting and manual adjustment to best fit into the electron density and satisfy chemical constraints. The data quality and refinement statistics are summarized in Table 2. The protein was thoroughly dialysed against 25 mM Pipes pH 7. The protein concentration was determined using the Bradford assay. Stock solutions of phloretin, chloramphenicol and naringenin at a concentration of mM were prepared in dimethyl sulfoxide and the solutions were subsequently diluted with dialysis buffer to final concentrations of 1 mM naringenin or 2 mM phloretin.

The corresponding amount of dimethyl sulfoxide was added to the protein sample. Each titration involved injections of effectors into protein solution. Identical experimental conditions were used to analyse wild-type as well as mutant proteins.

The mean enthalpies measured from injection of the ligands into the buffer were subtracted from raw titration data prior to data analysis with ORIGIN software MicroCal. Electrophoretic mobility shift assays were carried out as described. We also thank the Royal Society, which supported the collaborative visits between X. We are grateful to members in X. Appendix A Supplementary data associated with this article can be found, in the online version, at doi Effector molecules are displayed as stick models and colored in yellow.

Note the two phloretin molecules within the same pocket. The horizontal one has better defined electron density than the vertical one. Phloretin molecules are shown as sticks. Arg is labeled. Color scheme: yellow—carbon atoms in phloretin, red—oxygen atoms, blue- nitrogen atoms. In TtgR structure, two phloretin molecules are present in one binding pocket, one molecule lying horizontally while the second one vertically.

In TtgR RG structure, only one phloretin molecule is present lying in the vertical location. Sponsored Document from. J Mol Biol. Juan L. Author information Article notes Copyright and License information Disclaimer. Xiaodong Zhang: ku. This article has been cited by other articles in PMC. Abstract Antibiotic resistance is a widely spread phenomenon. Keywords: crystal structure, multidrug binding, antibiotic resistance, ITC, protein—ligand interaction.

Introduction Microorganisms are exposed to naturally occurring deleterious chemicals in the environment, like natural antibiotics produced by bacteria, fungi and plants, or detergents such as bile salts, present in the intestinal tract of higher animals. Results Overall TtgR structure Since TtgR crystals diffract poorly in the absence of its ligands, we therefore focus here on TtgR structures in complex with different ligands.

Open in a separate window. Figure 1. Figure 2. TtgR in complex with antibiotics TtgR can recognize a wide range of molecules including a number of antibiotics, flavonoids, as well as aromatic solvents. Figure 3. TtgR in complex with flavonoids Several flavonoids have antimicrobial properties, and indeed, their ability to bind to TtgR and induce the expression of the corresponding TtgABC efflux pump was recently demonstrated.

TtgR in complex with quercetin and naringenin Quercetin and naringenin have similar structures consisting of a chromenone ring and a hydroxyphenyl ring Figure 3 a. TtgR in complex with phloretin Phloretin contains a 2,4,6-trihydroxyphenyl ring connected to a 4-hydroxyphenyl ring. Figure 4. Mutagenesis studies to confirm the binding sites Arg plays an important role in phloretin binding at the high affinity site. Table 1 Thermodynamic parameters derived from the microcalorimetric titration of wild-type and mutant TtgR with different effector molecules.

Discussion Two distinct binding sites contribute to the versatile binding property of TtgR Our work has identified two distinct binding sites within the large pocket in TtgR Figure 1 c , which contributes to the triangular shape of the binding pocket. TtgR displays unique binding sites compared to other TetR family members Structurally TtgR is a member of the TetR family of transcriptional repressors.

Figure 5. Implications in multidrug resistance The data presented here explain how TtgR could recognize a wide range of antibiotics and plant secondary metabolites. Materials and Methods Protein expression, purification, and crystallisation TtgR proteins were expressed and purified as described. Table 2 Data collection and refinement statistics. Electrophoretic mobility shift assay Electrophoretic mobility shift assays were carried out as described.

Structural analysis and molecular graphics TtgR structures full-length, DNA binding domain, and ligand binding domain were submitted to DALI server 31 to obtain structural homologues. Notes Edited by I. Footnotes Appendix A Supplementary data associated with this article can be found, in the online version, at doi Appendix A. Supplementary data Supplementary Figure S1. Supplementary Figure S2. References 1. Nikaido H. Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria.

Cell Dev. Ramos J. Mechanisms of solvent tolerance in Gram-negative bacteria. Markham P. The drug-binding activity of the multidrug-responding transcriptional regulator BmrR resides in its C-terminal domain.

Brooun A. Purification and ligand binding of EmrR, a regulator of a multidrug transporter. Grkovic S. QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA. Teran W. Agents Chemother. Effector-repressor interactions, binding of a single effector molecule to the operator-bound TtgR homodimer mediates derepression. Espinosa-Urgel M. Root colonization by Pseudomonas putida : love at first sight.

Kuiper I. Selection of a plant-bacterium pair as a novel tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria. Plant Microbe Interact. Isolation and expansion of the catabolic potential of a Pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons. Rojas A. Duque E. Effector-repressor interactions. Binding of a single effector molecule to the operator bound TtgR homodimer mediates derepression.

Guazzaroni M. TtgV bound to a complex operator site represses transcription of the promoter for the multidrug and solvent extrusion TtgGHI pump. Dastidar S. Antimicrobial activity of prenylflavanones. In Vivo. Rauha J. Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds.

Food Microbiol. Plaper A. Characterization of quercetin binding site on DNA gyrase. The TetR family of transcriptional repressors. Hinrichs W. Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance. Schumacher M. Deciphering the molecular basis of multidrug recognition: crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR. Structural mechanisms of QacR induction and multidrug recognition.

Teran, W. Molecular characterization of its regulator TtgR. PhD, Universidad de Granada. Otwinowski Z. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. Leslie A. Hendrickson W. Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction MAD : a vehicle for direct determination of three-dimensional structure.

EMBO J. Terwilliger T. Maximum-likelihood density modification. Acta Crystallog. Jones T. Improved methods for building protein models in electron density maps and the location of errors in these models. Emsley P. Coot: model-building tools for molecular graphics. Brunger A. Select item s and click on "Add to basket" to create your own collection here entries max. Probable regulatory protein for the ttgABC efflux pump operon.

May function as a repressor By similarity. Manual assertion according to rules i. You are using a version of browser that may not display all the features of this website. Please consider upgrading your browser. We will be switching to the new UniProt website in a few weeks.

Please explore and share your feedback. Take me to the new website. Your basket is currently empty. Note that the 'protein existence' evidence does not give information on the accuracy or correctness of the sequence s displayed. Select a section on the left to see content.

By similarity. Alternative name s :. Name: ttgR. DrugCentral i Q88N ModBase i Search The information is filed in different subsections. Length: Mass Da : 23, It is useful for tracking sequence updates. The algorithm is described in the ISO standard. Pseudomonas sp. Pseudomonas putida S Full view. Pseudomonas monteilii. Pseudomonas juntendi. Pseudomonas plecoglossicida NB These are stable identifiers and should be used to cite UniProtKB entries.

Upon integration into UniProtKB, each entry is assigned a unique accession number, which is called 'Primary citable accession number'.

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