The molecular mechanisms of allosteric mutations impairing MepR repressor function in multidrug-resistant strains of Staphylococcus aureus.
The molecular mechanisms of allosteric mutations impairing MepR repressor function in multidrug-resistant strains of Staphylococcus aureus.
复制标题
DOI：
10.1128/mbio.00528-13
复制
发表时间：
2013-08-27
期刊：
影响因子：
6.4
通讯作者：
Brennan RG
中科院分区：
文献类型：
--
作者：
Birukou I;Tonthat NK;Seo SM;Schindler BD;Kaatz GW;Brennan RG
金黄色葡萄球菌多药外排泵MepA的过表达赋予对多种抗菌剂的抗性。mepA表达受马尔R家族成员MepR控制，MepR抑制mepA并自动抑制其自身的产生。mepR突变是多重耐药金黄色葡萄球菌临床分离株中mepA过表达的主要原因。在这里，我们报告了三种多药耐药MepR变体的晶体结构，这些变体包含单氨基酸取代A103 V，F27 L或Q18 P，以及野生型MepR的DNA结合构象。虽然每个突变通过降低其DNA结合亲和力来损害MepR功能，但是没有一个突变位于DNA结合结构域中。相反，所有这些都存在于连接二聚化和DNA结合结构域的接头区中。具体而言，A103 V取代作用于F27，F27通过置换DNA结合翼-螺旋-转角-螺旋基序解决潜在的空间冲突，导致DNA结合亲和力降低27倍。F27 L取代迫使F104成为另一种旋转异构体，其使螺旋5扭结，从而干扰DNA结合结构域的定位并使mepR操纵基因亲和力降低35倍。Q18 P突变通过在螺旋1的中间产生扭结或完全展开其C末端来最显著地影响MepR结构和功能。此外，Q18 P的螺旋5被弯曲或完全切割成两个较小的螺旋。因此，DNA结合减少了2,000倍。我们的结构研究揭示了迄今未观察到的影响马尔R家族成员阻遏物功能并导致多重耐药金黄色葡萄球菌的变构机制。金黄色葡萄球菌是免疫功能低下患者的主要健康威胁。过表达多药外排泵mepA的金黄色葡萄球菌多药耐药变异体经常出现，这是由于mepA转录阻遏物马尔R家族成员MepR中的点突变。值得注意的是，在这些金黄色葡萄球菌临床分离株中鉴定的大多数MepR突变不是在DNA结合结构域中发现的，而是在连接二聚化和DNA结合结构域的接头区中发现的。这些突变体的位置强调了调节MepR功能的正常功能的变构机制的至关重要性。理解这种变构MepR突变体的失调是这项研究的基础。三个这样的变构MepR突变体的高分辨率结构揭示了不可预测的构象后果，所有这些都排除了同源DNA结合，而生物化学研究强调了它们对DNA结合亲和力的削弱作用。因此，MepR连接区的突变及其结构后果是多重耐药金黄色葡萄球菌的关键发生器。
Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus. Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.