Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern
Structural basis for the arsenite binding and translocation of Acr3 antiporter with NhaA folding pattern
复制标题
具有 NhaA 折叠模式的 Acr3 反向转运蛋白的亚砷酸盐结合和易位的结构基础
DOI：
10.1096/fj.202201280r
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Chunyu Yang
中科院分区：
文献类型：
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作者：
Peiwen Lv;Yan Shang;Ye Zhang;Wenkai Wang;Yuanxiang Liu;D;an Su;Wei Wang;Chunfang Li;Cuiqing Ma;Chunyu Yang
砷抗性-3(ACR3)家族是在各种微生物和低等植物中对有毒金属类化合物产生高水平抗性的最常见的途径。根据AlphaFold2构建的结构模型，枯草芽孢杆菌Acr3反向转运蛋白(Acr3BS)呈现典型的NhaA结构折叠，TM4和TM9两个不连续的跨膜片段螺旋相互作用，形成X形结构。由于这些重要的砷外排泵的结构信息有限，我们研究了300个同源序列之间的进化保守性，并在不连续的螺旋和TM5中发现了三个保守的基序。通过定点突变、微尺度热电泳法(MST)和荧光共振能量转移(FRET)分析，发现TM9中的基序C是底物结合的关键元件，其中N292和e295参与底物配位，而TM4中的R118和TM10中的E322负责结构稳定。此外，TM5基序B上的高度保守残基是质子化/去质子化过程中的潜在关键因素。这些共有的基序和残基是类金属化合物转运Acr3逆向转运蛋白所必需的，它们构筑了核心区和典型的NhaA折叠的X形结构。
The arsenical resistance‐3 (ACR3) family constitutes the most common pathway that confers high‐level resistance to toxic metalloids in various microorganisms and lower plants. Based on the structural model constructed by AlphaFold2, the Acr3 antiporter fromBacillus subtilis(Acr3Bs) exhibits a typical NhaA structure fold, with two discontinuous helices of transmembrane (TM) segments, TM4 and TM9, interacting with each other and forming an X‐shaped structure. As the structural information available for these important arsenite‐efflux pumps is limited, we investigated the evolutionary conservation among 300 homolog sequences and identified three conserved motifs in both the discontinuous helices and TM5. Through site‐directed mutagenesis, microscale thermophoresis (MST), and fluorescence resonance energy transfer (FRET) analyses, the identified Motif C in TM9 was found to be a critical element for substrate binding, in which N292 and E295 are involved in substrate coordination, while R118 in TM4 and E322 in TM10 is responsible for structural stabilization. In addition, the highly conserved residues on Motif B of TM5 are potentially key factors in the protonation/deprotonation process. These consensus motifs and residues are essential for metalloid compound translocation of Acr3 antiporters, by framing the core domain and the typical X‐shaped of NhaA fold.