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.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Peters KM,Sharbeen G,Theis T,Skurray RA,Brown MHdoi
10.1021/bi901102hsubject
Has Abstractpub_date
2009-10-20 00:00:00pages
9794-800issue
41eissn
0006-2960issn
1520-4995journal_volume
48pub_type
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