Evolutionary conservative analysis revealed novel functional sites in the efflux pump NorA of Staphylococcus aureus
Evolutionary conservative analysis revealed novel functional sites in the efflux pump NorA of Staphylococcus aureus
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进化保守分析揭示金黄色葡萄球菌外排泵 NorA 的新功能位点
DOI：
10.1093/jac/dkab453
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发表时间：
2021
影响因子：
5.2
通讯作者：
Chunyu Yang
中科院分区：
文献类型：
--
作者：
Yan Shang;Peiwen Lv;D;an Su;Yaru Li;Yu Liang;Cuiqing Ma;Chunyu Yang
抽象的。目标。金黄色葡萄球菌的NorA逆向转运蛋白属于主要促进子超家族(MFS)，可分泌多种药物。由于该药物外排泵没有可用的结构，本研究的目的是探索其有助于底物结合和药物转运的重要结构元件。方法。对 NorA 同源物的不同编译进行进化保守性分析，以鉴定保守基序和残基。构建定点突变以验证 NorA 流出能力的功能变化，并通过荧光共振能量转移 (FRET) 和微尺度热泳 (MST) 分析进一步测量构象变化。结果。除了之前在 MFS 蛋白中报道的 Motif-A、Motif-B 和 Motif-C 之外，在 NorA 中还鉴定出了另外两个基序：Motif-1 和 Motif-2。 Motif-1和Motif-2的定点突变以及11个预测的结合位点均导致耐药性和外排活性显着降低。其中，通过MST测量，突变体F16A/E222A/F303A/D307A对四苯基氯化鏻的结合亲和力发生改变，Motif-1突变体G114D/A117E/D118G/V119I和Motif-2突变体Q325E/G326E/A328E/G330E与突变体相比表现出明显的构象改变。 FRET 信号光谱中的野生型 NorA。结论。 NorA结构与MFS蛋白的典型结构非常吻合，有两个新发现的基序（Motif-1和Motif-2）对NorA的结构稳定性至关重要，位点F16、E222、F303和D307参与底物结合。
Abstract. Objectives. The NorA antiporter of Staphylococcus aureus belongs to the major facilitator superfamily (MFS) and extrudes various kinds of drugs. With no structure available for this drug efflux pump, the aim of this study was to explore its important structural elements that contribute to substrate binding and drug transport.. Methods. Evolutionary conservative analyses were conducted on different compilations of NorA homologues to identify conservative motifs and residues. Site-directed mutations were constructed to verify the functional changes in NorA efflux capacities and the conformational changes were further measured by fluorescence resonance energy transfer (FRET) and microscale thermophoresis (MST) analysis.. Results. Besides Motif-A, Motif-B and Motif-C that were reported previously in MFS proteins, two other motifs, Motif-1 and Motif-2, were identified in NorA. Site-directed mutations of Motif-1 and Motif-2 as well as 11 predicted binding sites all caused remarkable reductions in drug resistance and efflux activity. Among these, mutant F16A/E222A/F303A/D307A showed an altered binding affinity for tetraphenylphosphonium chloride when measured by MST and Motif-1 mutant G114D/A117E/D118G/V119I and Motif-2 mutant Q325E/G326E/A328E/G330E displayed obvious conformational alterations when compared with the wild-type NorA in the FRET signal spectra.. Conclusions. The NorA structure agrees well with the typical structures of MFS proteins, with two newly identified motifs (Motif-1 and Motif-2) that are critical to the structural stability of NorA, and sites F16, E222, F303 and D307 are involved in substrate binding.