Interactions of the QacR Multidrug-Binding Protein with Structurally Diverse Ligands:  Implications for the Evolution of the Binding Pocket

Grkovic, Steve; Hardie, Kate M; Brown, Melissa H.; Skurray, Ronald A.

doi:10.1021/bi035447+

Cited by 74 publications

(75 citation statements)

References 39 publications

(86 reference statements)

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“…The affinities of AcrR to its drugs are in the micromolar region, and the dissociation constants are quite similar to those of QacR [25], BmrR [26], and TtgV [27]. Based on the fluorescence polarization assays, titrations of AcrR to the ligand binding solutions give a stoichiometry of 1:1 monomeric AcrR-to-drug molar ratio.…”

Section: Discussionmentioning

confidence: 83%

Characterization of the multidrug efflux regulator AcrR from Escherichia coli

Su¹,

Rutherford²,

Yu³

2007

Biochemical and Biophysical Research Communications

101

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The Escherichia coli AcrR represses transcription of the acrB gene, which encodes the multidrug efflux pump AcrB that extrudes a wide variety of toxic compounds, by binding its target operator DNA. Fluorescence polarization was performed using purified, recombinant AcrR that contains a 6xHis tag at the C-terminus and a fluorescein-labeled 28-base pair oligonucleotide bearing a predicted palindrome (IR) operator sequence. Binding of AcrR to the predicted IR sequence occurred with a dissociation constant (K D ) in the nanomolar range. Fluorescence polarization assays were also applied to characterize the affinity and specificity of AcrR interaction with three different fluorescent ligands, rhodamine 6G, ethidium and proflavin. The K D values for these ligands range from 4.2 to 10.1 μM, suggesting that AcrR is capable of recognizing a wide range of structurally dissimilar toxic compounds as it is in the case of the AcrB multidrug efflux pump. We found that the binding of rhodamine 6G to AcrR is inhibited by the presence of ethidium. In contrast, the dissociation constant of proflavin binding to AcrR was not affected by ethidium, a result suggesting that ethidium and proflavin are bound to distinct binding sites.

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Section: Discussionmentioning

confidence: 83%

Characterization of the multidrug efflux regulator AcrR from Escherichia coli

Su¹,

Rutherford²,

Yu³

2007

Biochemical and Biophysical Research Communications

101

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“…It has been suggested that binding of the first QacR dimer forces this energetically unfavorable conformational change, which in turn produces an optimal DNA conformation for the easy binding of the second dimer (351). Experimental data reported by Grkovic et al (121,122) suggested that the two dimers must bind simultaneously and cooperatively to the operator in order to maintain the DNA deformation detected in the crystal.…”

Section: Qacr Regulatormentioning

confidence: 99%

“…QacR is released from the qacA operator by its interaction with a number of cationic lipophilic drugs such as rhodamine 6G, crystal violet, and ethidium (119). More recently, Grkovic et al (122) showed that effector recognition of QacR can be extended to several bivalent cationic dyes and plant alkaloids. In spite of the existence of two binding pockets, only one drug molecule is bound by each homodimer, as determined by equilibrium dialysis studies and isothermal titration calorimetry for the QacR-R6G complex (350).…”

Section: Downloaded Frommentioning

confidence: 99%

“…The following color code was used for complex networks: dark blue, TetR family member; orange, the gene directly regulated by the TetR family member; light blue, a regulator that modulates the expression of a TetR family member or which assists in the regulation of the gene under the control of a TetR family member; yellow boxes, signals and conditions influencing the system; open boxes, final results of the action of the system when the result is a scorable phenotype. References recommended for each circuit: panel A (29,38,286,288,388,417,418); panel B (4,7,13,31,35,110,176,191,230,245,270,436); panel C (8, 91, 201-204, 222, 290, 331); panel D (119,120,122,249,261,332,(350)(351); panel E (5,6,66,67,116,163); panel F (228,238,397,333,353,408); panel G (87-89, 125, 140, 214, 215, 295, 360, 403); panel H (48, 161); panel I (78); panel J (56,184,185,…”

Section: Beti Controls the Choline-glycine Betaine Pathway Of E Colimentioning

confidence: 99%

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The TetR Family of Transcriptional Repressors

Ramos

Martínez-Bueno

Molina‐Henares

et al. 2005

Microbiol Mol Biol Rev

990

1,133

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We have developed a general profile for the proteins of the TetR family of repressors. The stretch that best defines the profile of this family is made up of 47 amino acid residues that correspond to the helix-turn-helix DNA binding motif and adjacent regions in the three-dimensional structures of TetR, QacR, CprB, and EthR, four family members for which the function and three-dimensional structure are known. We have detected a set of 2,353 nonredundant proteins belonging to this family by screening genome and protein databases with the TetR profile. Proteins of the TetR family have been found in 115 genera of gram-positive, α-, β-, and γ-proteobacteria, cyanobacteria, and archaea. The set of genes they regulate is known for 85 out of the 2,353 members of the family. These proteins are involved in the transcriptional control of multidrug efflux pumps, pathways for the biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, control of catabolic pathways, differentiation processes, and pathogenicity. The regulatory network in which the family member is involved can be simple, as in TetR (i.e., TetR bound to the target operator represses tetA transcription and is released in the presence of tetracycline), or more complex, involving a series of regulatory cascades in which either the expression of the TetR family member is modulated by another regulator or the TetR family member triggers a cell response to react to environmental insults. Based on what has been learned from the cocrystals of TetR and QacR with their target operators and from their three-dimensional structures in the absence and in the presence of ligands, and based on multialignment analyses of the conserved stretch of 47 amino acids in the 2,353 TetR family members, two groups of residues have been identified. One group includes highly conserved positions involved in the proper orientation of the helix-turn-helix motif and hence seems to play a structural role. The other set of less conserved residues are involved in establishing contacts with the phosphate backbone and target bases in the operator. Information related to the TetR family of regulators has been updated in a database that can be accessed at www.bactregulators.org

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“…Like LfrR, the majority of QacR-ligand structures show a single ligand within one monomer of each dimer. However, one structure of QacR in complex with two different ligands, ethidium and proflavine, within the same monomer has been solved (60). For CgmR, different binding stoichiometries are seen for different drugs, and the size of the drug is thought to play a role in the number of molecules required for CgmR derepression (43).…”

Section: Tfr-ligand Interactionsmentioning

confidence: 99%

The TetR Family of Regulators

Cuthbertson

Nodwell

2013

Microbiol Mol Biol Rev

480

512

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SUMMARY The most common prokaryotic signal transduction mechanisms are the one-component systems in which a single polypeptide contains both a sensory domain and a DNA-binding domain. Among the >20 classes of one-component systems, the TetR family of regulators (TFRs) are widely associated with antibiotic resistance and the regulation of genes encoding small-molecule exporters. However, TFRs play a much broader role, controlling genes involved in metabolism, antibiotic production, quorum sensing, and many other aspects of prokaryotic physiology. There are several well-established model systems for understanding these important proteins, and structural studies have begun to unveil the mechanisms by which they bind DNA and recognize small-molecule ligands. The sequences for more than 200,000 TFRs are available in the public databases, and genomics studies are identifying their target genes. Three-dimensional structures have been solved for close to 200 TFRs. Comparison of these structures reveals a common overall architecture of nine conserved α helices. The most important open question concerning TFR biology is the nature and diversity of their ligands and how these relate to the biochemical processes under their control.

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Interactions of the QacR Multidrug-Binding Protein with Structurally Diverse Ligands: Implications for the Evolution of the Binding Pocket

Cited by 74 publications

References 39 publications

Characterization of the multidrug efflux regulator AcrR from Escherichia coli

Characterization of the multidrug efflux regulator AcrR from Escherichia coli

The TetR Family of Transcriptional Repressors

The TetR Family of Regulators

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