Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
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DOI：
10.1002/pro.343
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发表时间：
2010-04-01
期刊：
影响因子：
8
通讯作者：
Sunnerhagen, Maria
中科院分区：
文献类型：
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作者：
Andresen, Cecilia;Jalal, Shah;Sunnerhagen, Maria
由mexO操纵子编码的自组装MexA-MexB-OprM外排泵系统通过主动挤出多种抗菌剂而有助于铜绿假单胞菌的简易耐药性。MexR负调节mexO操纵子，包括两个相邻的MexR结合位点，并且因此被赋予多药耐药性（MDR）的突变高度靶向。为了了解MDR突变如何损害MexR功能，我们研究了MexR-wt以及一组选定的MDR单突变体远离拟议的DNA结合螺旋。尽管在所选的MexR-MDR突变体中DNA亲和力和MexA-MexB-OprM阻遏都显著受损，但MexR-wt作为二聚体以高亲和力结合其在mexO中的两个结合位点。在MexR-MDR突变体中，二级结构含量和寡聚化性质与MexR-wt非常相似，尽管它们缺乏DNA结合。尽管如此，MexR-MDR突变体表现出高度不同的稳定性相比，MexR-wt，这表明干扰的关键域间接触，因为在DNA结合结构域的突变影响的二聚体区域的稳定性，反之亦然。此外，显着的ANS结合MexR-wt在自由和DNA结合状态，以及增加ANS结合在所有研究的突变体，表明在二聚体区域中的疏水腔已经被证明参与调节结合扩大MDR突变。两者合计，我们建议，生物物理MexR属性，MDR突变的稳定性，结构域相互作用，和内部疏水表面的目标也是至关重要的MexR DNA结合的调节。
The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.