Biochemical characterization of the multidrug regulator QacR distinguishes residues that are crucial to multidrug binding and induction of qacA transcription.

Full Abstract

Staphylococcus aureus transcription factor QacR regulates expression of the qacA multidrug efflux determinant. In response to binding cationic lipophilic compounds, including ethidium and rhodamine 6G, QacR dissociates from the qacA operator alleviating repression. Such ligand binding uniformly induces a coil-to-helix transition of residues Thr(89)-Tyr(93) revealing an asymmetric binding pocket in QacR containing two distinct subpockets. Here, the functional significance of hydrophobic, aromatic, and polar residues characteristic of the rhodamine 6G pocket and the proximal Tyr(92), proposed to facilitate the transcriptionally active conformation, was examined. Notably, the presence of Tyr(92) was not essential for QacR structural changes between DNA-bound and induced conformations. Furthermore, although mutation of the majority of residues contacting rhodamine 6G exerted moderate effects on QacR-rhodamine 6G binding, mutation of Leu(54) and Gln(96), and cumulative mutations involving these with Tyr(93) and Tyr(123), imparted a dramatic decrease in QacR-rhodamine 6G binding affinity. This equated with impaired dissociation of QacR from its operator DNA in the presence of this ligand in S. aureus, delineating the important role of these residues in the QacR-rhodamine 6G interaction. Additionally, despite maintaining a high affinity for ethidium, QacR mutants involving Leu(54), Tyr(93), Gln(96), and Tyr(123), which denote the interface between the rhodamine 6G and ethidium subpockets, were unable to be induced from operator DNA in the presence of ethidium in S. aureus. This highlights the significant contribution of these residues to QacR-mediated derepression of qacA transcription following ligand binding in the distal subpocket and may be important for the general mechanism irrespective of the ligand bound.

Author information

Author/s: Peters, Kate M (KM); Sharbeen, George (G); Theis, Torsten (T); Skurray, Ronald A (RA); Brown, Melissa H (MH);

Affiliation: School of Biological Sciences, A12, University of Sydney, Sydney, NSW, Australia.

Journal and publication information

Publication Type: Journal Article; Research Support, Non-U.S. Gov't

Journal: Biochemistry (Biochemistry), published in United States. (Language: eng)

Reference: 2009-Oct; vol 48 (issue 41) : pp 9794-800

Dates: Created 2009/10/13; Completed 2009/11/02;

PMID: 19761200, status: MEDLINE (last retrieval date: 11/2/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Amino Acid Substitution
Bacterial Proteins - chemistry; genetics; metabolism
Binding Sites
Drug Resistance, Microbial
Ethidium - metabolism
Homeostasis
Ligands
Membrane Transport Proteins - genetics

Membrane Transport Proteins - genetics
Models, Molecular
Mutation
Protein Conformation
Repressor Proteins - chemistry; genetics; metabolism
Rhodamines - metabolism
Staphylococcus aureus - genetics
Transcription, Genetic

Associated Chemicals: Bacterial Proteins (0) ; Ligands (0) ; Membrane Transport Proteins (0) ; QacR protein, Staphylococcus aureus (0) ; Repressor Proteins (0) ; Rhodamines (0) ; qacA protein, Staphylococcus aureus (134773-66-3) ; Ethidium (3546-21-2)

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