Crystal Structures of QacR-Diamidine Complexes Reveal Additional Multidrug-binding Modes and a Novel Mechanism of Drug Charge Neutralization

Murray, David S.; Schumacher, Maria A.; Brennan, Richard G.

doi:10.1074/jbc.m313870200

Cited by 75 publications

(107 citation statements)

References 31 publications

(36 reference statements)

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“…4). A similar duality (complementary between hydrophobic and polar interactions) was observed for the binding of effector molecules to other multidrug binding regulators such as BmrR (22) and QacR (23,24) and might represent a general feature for multidrug recognition by this class of proteins.…”

Section: Discussionmentioning

confidence: 81%

“…Moreover, a number of studies have shown that the expression of some MDR transporters is controlled by transcriptional regulators with the same multidrug recognition properties, such as BmrR of Bacillus subtilis (18), EmrR of E. coli (19), QacR of Staphylococcus aureus (20), and TtgR of Pseudomonas putida DOT-T1E (21). In that sense, the recent determination of the three-dimensional structures of QacR and BmrR in complex with several ligands has provided important insight into the molecular mechanism of multiple drug recognition (22)(23)(24).…”

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confidence: 99%

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Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression

Terán¹,

Krell

Ramos

et al. 2006

Journal of Biological Chemistry

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The RND family transporter TtgABC and its cognate repressor TtgR from Pseudomonas putida DOT-T1E were both shown to possess multidrug recognition properties. Structurally unrelated molecules such as chloramphenicol, butyl paraben, 1,3-dihydroxynaphthalene, and several flavonoids are substrates of TtgABC and activate pump expression by binding to the TtgR-operator complex. Isothermal titration calorimetry was employed to determine the thermodynamic parameters for the binding of these molecules to TtgR. Dissociation constants were in the range from 1 to 150 M, the binding stoichiometry was one effector molecule per dimer of TtgR, and the process was driven by favorable enthalpy changes. Although TtgR exhibits a large multidrug binding profile, the plantderived compounds phloretin and quercetin were shown to bind with the highest affinity (K D of around 1 M), in contrast to other effectors (chloramphenicol and aromatic solvents) for which exhibited a more reduced affinity. Structure-function studies of effectors indicate that the presence of aromatic rings as well as hydroxyl groups are determinants for TtgR binding. The binding of TtgR to its operator DNA does not alter the protein effector profile nor the effector binding stoichiometry. Moreover, we demonstrate here for the first time that the binding of a single effector molecule to the DNA-bound TtgR homodimer induces the dissociation of the repressor-operator complex. This provides important insight into the molecular mechanism of effector-mediated derepression.

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Section: Discussionmentioning

confidence: 81%

mentioning

confidence: 99%

Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression

Terán¹,

Krell

Ramos

et al. 2006

Journal of Biological Chemistry

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“…Similar drug-regulator interaction has been found in QacR, in which the QacR regulator neutralized one end of the positively charged pentamidine by using carbonyl and side chain oxygen atoms. 27 Interestingly, the bound Chd rather employed another mechanism to neutralize its formal negative charge, whereas the anionic pentanoate group was compensated by the formal positive charge of H174.…”

Section: Discussionmentioning

confidence: 99%

Crystal structures of CmeR‐bile acid complexes from Campylobacter jejuni

et al. 2011

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The TetR family of transcription regulators are diverse proteins capable of sensing and responding to various structurally dissimilar antimicrobial agents. Upon detecting these agents, the regulators allow transcription of an appropriate array of resistance markers to counteract the deleterious compounds. Campylobacter jejuni CmeR is a pleiotropic regulator of multiple proteins, including the membrane-bound multidrug efflux transporter CmeABC. CmeR represses the expression of CmeABC and is induced by bile acids, which are substrates of the CmeABC tripartite pump. The multiligand-binding pocket of CmeR has been shown to be very extensive and consists of several positively charged and multiple aromatic amino acids. Here we describe the crystal structures of CmeR in complexes with the bile acids, taurocholate and cholate. Taurocholate and cholate are structurally related, differing by only the anionic charged group. However, these two ligands bind distinctly in the binding tunnel. Taurocholate spans the novel bile acid binding site adjacent to and without overlapping with the previously determined glycerol-binding site. The anionic aminoethanesulfonate group of taurocholate is neutralized by a charge-dipole interaction. Unlike taurocholate, cholate binds in an anti-parallel orientation but occupies the same bile acidbinding site. Its anionic pentanoate moiety makes a water-mediated hydrogen bond with a cationic residue to neutralize the formal negative charge. These structures underscore the promiscuity of the multifaceted binding pocket of CmeR. The capacity of CmeR to recognize bile acids was confirmed using isothermal titration calorimetry and fluorescence polarization. The results revealed that the regulator binds these acids with dissociation constants in the micromolar region.

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“…Gly-313 has also been identified as a functionally important residue that appears to assume a structurally important role common to the transport process. Furthermore, mutagenic studies clearly demonstrate the multifaceted nature and flexibility of the multidrugbinding sites that can easily adapt to modifications and that appear to be a common trend among multidrug-binding proteins (42)(43)(44)(45)(46). …”

Section: Discussionmentioning

confidence: 99%

Role of Transmembrane Segment 10 in Efflux Mediated by the Staphylococcal Multidrug Transport Protein QacA

O’Rourke²,

Skurray

et al. 2006

Journal of Biological Chemistry

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The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4,6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrugbinding pocket.

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Crystal Structures of QacR-Diamidine Complexes Reveal Additional Multidrug-binding Modes and a Novel Mechanism of Drug Charge Neutralization

Cited by 75 publications

References 31 publications

Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression

Effector-Repressor Interactions, Binding of a Single Effector Molecule to the Operator-bound TtgR Homodimer Mediates Derepression

Crystal structures of CmeR‐bile acid complexes from Campylobacter jejuni

Role of Transmembrane Segment 10 in Efflux Mediated by the Staphylococcal Multidrug Transport Protein QacA

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