A key mechanism that Neisseria gonorrhoeae uses to achieve multidrug resistance is the expulsion of structurally different antimicrobials by the MtrD multidrug efflux protein. MtrD resembles the homologous Escherichia coli AcrB efflux protein with several common structural features, including an open cleft containing putative access and deep binding pockets proposed to interact with substrates. A highly discriminating N. gonorrhoeae strain, with the MtrD and NorM multidrug efflux pumps inactivated, was constructed and used to confirm and extend the substrate profile of MtrD to include 14 new compounds. The structural basis of substrate interactions with MtrD was interrogated by a combination of long-timescale molecular dynamics simulations and docking studies together with site-directed mutagenesis of selected residues. Of the MtrD mutants generated, only one (S611A) retained a wild-type (WT) resistance profile, while others (F136A, F176A, I605A, F610A, F612C, and F623C) showed reduced resistance to different antimicrobial compounds. Docking studies of eight MtrD substrates confirmed that many of the mutated residues play important nonspecific roles in binding to these substrates. Long-timescale molecular dynamics simulations of MtrD with its substrate progesterone showed the spontaneous binding of the substrate to the access pocket of the binding cleft and its subsequent penetration into the deep binding pocket, allowing the permeation pathway for a substrate through this important resistance mechanism to be identified. These findings provide a detailed picture of the interaction of MtrD with substrates that can be used as a basis for rational antibiotic and inhibitor design.IMPORTANCE With over 78 million new infections globally each year, gonorrhea remains a frustratingly common infection. Continuous development and spread of antimicrobial-resistant strains of Neisseria gonorrhoeae, the causative agent of gonorrhea, have posed a serious threat to public health. One of the mechanisms in N. gonorrhoeae involved in resistance to multiple drugs is performed by the MtrD multidrug resistance efflux pump. This study demonstrated that the MtrD pump has a broader substrate specificity than previously proposed and identified a cluster of residues important for drug binding and translocation. Additionally, a permeation pathway for the MtrD substrate progesterone actively moving through the protein was determined, revealing key interactions within the putative MtrD drug binding pockets. Identification of functionally important residues and substrate-protein interactions of the MtrD protein is crucial to develop future strategies for the treatment of multidrug-resistant gonorrhea.

中文翻译：

---

淋病奈瑟氏球菌的多药耐药性：MtrD外排蛋白中功能上重要的残留物的鉴定。

淋病奈瑟氏球菌用于实现多药耐药性的关键机制是MtrD多药外排蛋白排出结构上不同的抗菌素。MtrD类似于具有多个常见结构特征的同源大肠杆菌AcrB外排蛋白，包括一个开放的裂缝，该裂缝包含推定的通道和拟与底物相互作用的深层结合袋。构建了一个高度区分的淋病奈瑟氏球菌菌株，并停用了MtrD和NorM多药外排泵，并将其用于确认和扩展MtrD的底物谱，包括14种新化合物。长期分子动力学模拟和对接研究以及对选定残基的定点诱变相结合，对底物与MtrD相互作用的结构基础进行了研究。在生成的MtrD突变体中，只有一个（S611A）保留了野生型（WT）耐药性，而其他（F136A，F176A，I605A，F610A，F612C和F623C）表现出对不同抗菌化合物的耐药性降低。对八种MtrD底物的对接研究证实，许多突变残基在与这些底物结合中起重要的非特异性作用。MtrD及其底物孕酮的长期分子动力学模拟显示，底物自发结合到结合裂口的进入袋中，随后渗透到深层结合袋中，从而使底物透过这种重要的抗性机制渗透被识别。这些发现为MtrD与底物之间的相互作用提供了详细的图片，可作为合理的抗生素和抑制剂设计的基础。重要信息淋病每年在全球范围内有7800万以上的新感染，仍然是令人沮丧的常见感染。淋病奈瑟氏球菌的抗药性菌株的持续发展和传播已经严重威胁公共卫生。淋病奈瑟菌中涉及多种药物耐药性的机制之一是由MtrD多药耐药性外排泵完成的。这项研究表明，MtrD泵比以前提出的泵具有更宽的底物特异性，并鉴定了对药物结合和转运很重要的残基簇。另外，确定了MtrD底物孕酮主动移动通过蛋白质的渗透途径，揭示了推定的MtrD药物结合口袋中的关键相互作用。鉴定功能重要的残基和MtrD蛋白的底物-蛋白相互作用对于开发治疗多重耐药性淋病的未来策略至关重要。