The Staphylococcal QacR Multidrug Regulator Binds a Correctly Spaced Operator as a Pair of Dimers

Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.

金黄色葡萄球菌（Staphylococcus aureus）质粒编码的QacA多药转运蛋白的表达，受反向编码的QacR阻遏蛋白调控。为规避二硫键交联降解产物的形成，研究人员针对野生型QacR的两个半胱氨酸残基实施了定点诱变（site-directed mutagenesis）替换操作。对所得无半胱氨酸QacR变体的分析表明，该变体在体内（in vivo）与体外（in vitro）均保留完整的DNA结合活性，且仍可完全介导qacA的表达诱导过程，以响应一系列结构迥异的多药转运蛋白底物。研究人员利用该无半胱氨酸QacR蛋白开展交联（cross-linking）实验与动态光散射（dynamic light-scattering）实验，结果证实其天然存在形式为二聚体；而凝胶过滤层析（gel filtration）实验则显示，每个DNA操纵位点可结合4个QacR分子。加入诱导化合物后，结合于操纵位点的4个QacR分子会以二聚体形式从DNA上解离。研究还发现，QacR二聚体与DNA的结合依赖于操纵子半位点（operator half-sites）的正确间距。针对QacR调控qacA表达的修正模型提出了一项非常规特征：该调控蛋白的一个二聚体可结合至每个操纵子半位点，该过程似乎无需QacR预先自组装为四聚体。