The ATPase SecA is an essential component of the bacterial Sec machinery, which transports proteins across the cytoplasmic membrane. Most SecA proteins contain a long C-terminal tail (CTT). In Escherichia coli, the CTT contains a structurally flexible linker domain and a small metal-binding domain (MBD). The MBD coordinates zinc via a conserved cysteine-containing motif and binds to SecB and ribosomes. In this study, we screened a high-density transposon library for mutants that affect the susceptibility of E. coli to sodium azide, which inhibits SecA-mediated translocation. Results from sequencing this library suggested that mutations removing the CTT make E. coli less susceptible to sodium azide at subinhibitory concentrations. Copurification experiments suggested that the MBD binds to iron and that azide disrupts iron binding. Azide also disrupted binding of SecA to membranes. Two other E. coli proteins that contain SecA-like MBDs, YecA and YchJ, also copurified with iron, and NMR spectroscopy experiments indicated that YecA binds iron via its MBD. Competition experiments and equilibrium binding measurements indicated that the SecA MBD binds preferentially to iron and that a conserved serine is required for this specificity. Finally, structural modeling suggested a plausible model for the octahedral coordination of iron. Taken together, our results suggest that SecA-like MBDs likely bind to iron in vivo.

中文翻译：

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铁是体内 SecA 样金属结合结构域的配体。

ATPase SecA 是细菌 Sec 机器的重要组成部分，负责跨细胞质膜运输蛋白质。大多数 SecA 蛋白含有长 C 端尾 (CTT)。在大肠杆菌中，CTT 包含一个结构灵活的接头结构域和一个小的金属结合结构域 (MBD)。MBD 通过保守的含半胱氨酸基序协调锌，并与 SecB 和核糖体结合。在这项研究中，我们筛选了一个高密度转座子库，寻找影响大肠杆菌对叠氮化钠敏感性的突变体，叠氮化钠抑制 SecA 介导的易位。该文库的测序结果表明，消除 CTT 的突变使大肠杆菌对亚抑制浓度的叠氮化钠不太敏感。共纯化实验表明，MBD 与铁结合，而叠氮化物会破坏铁结合。叠氮化物还破坏了 SecA 与膜的结合。另外两种含有 SecA 样 MBD 的大肠杆菌蛋白 YecA 和 YchJ 也与铁共纯化，NMR 波谱实验表明 YecA 通过其 MBD 结合铁。竞争实验和平衡结合测量表明 SecA MBD 优先与铁结合，并且这种特异性需要保守的丝氨酸。最后，结构建模提出了铁八面体配位的合理模型。综上所述，我们的结果表明 SecA 样 MBD 可能在体内与铁结合。竞争实验和平衡结合测量表明 SecA MBD 优先与铁结合，并且这种特异性需要保守的丝氨酸。最后，结构建模提出了铁八面体配位的合理模型。综上所述，我们的结果表明 SecA 样 MBD 可能在体内与铁结合。竞争实验和平衡结合测量表明 SecA MBD 优先与铁结合，并且这种特异性需要保守的丝氨酸。最后，结构建模提出了铁八面体配位的合理模型。综上所述，我们的结果表明 SecA 样 MBD 可能在体内与铁结合。