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Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

ABSTRACT: Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors.

SUBMITTER: Carette X

PROVIDER: S-EPMC3326297 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Carette Xavier X Blondiaux Nicolas N Willery Eve E Hoos Sylviane S Lecat-Guillet Nathalie N Lens Zoé Z Wohlkönig Alexandre A Wintjens René R Soror Sameh H SH Frénois Frédéric F Dirié Bertrand B Villeret Vincent V England Patrick P Lippens Guy G Deprez Benoit B Locht Camille C Willand Nicolas N Baulard Alain R AR

Nucleic acids research 20111209 7

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of Et ...[more]

PMID: 22156370

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Project description:Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (?(v)?(3) and ?(5)?(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for ?(v)?(3) and ?(5)?(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both ?(v)?(3) and ?(5)?(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, ?(v)?(3) sequesters strongly into raft-like domains and ?(5)?(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.

Dataset Information

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Publications

Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands.

Similar Datasets

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