Biochemical Characterization of the Multidrug Regulator QacR Distinguishes Residues That Are Crucial to Multidrug Binding and Induction of qacA Transcription

详细信息    查看全文

• 作者：Kate M. Peters ; George Sharbeen ; Torsten Theis ; Ronald A. Skurray ; Melissa H. Brown
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：October 20, 2009
• 年：2009
• 卷：48
• 期：41
• 页码：9794-9800
• 全文大小：848K
• 年卷期：v.48,no.41(October 20, 2009)
• ISSN：1520-4995

文摘

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 Thr⁸⁹−Tyr⁹³ 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 Tyr⁹², proposed to facilitate the transcriptionally active conformation, was examined. Notably, the presence of Tyr⁹² 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 Leu⁵⁴ and Gln⁹⁶, and cumulative mutations involving these with Tyr⁹³ and Tyr¹²³, 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 Leu⁵⁴, Tyr⁹³, Gln⁹⁶, and Tyr¹²³, 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.