School of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom.
Warwick Medical School, University of Warwick, Coventry, CV4 7AL, United Kingdom.
Proc Natl Acad Sci U S A. 2020 Nov 10;117(45):28355-28365. doi: 10.1073/pnas.2017134117. Epub 2020 Oct 23.
FtsEX is a bacterial ABC transporter that regulates the activity of periplasmic peptidoglycan amidases via its interaction with the murein hydrolase activator, EnvC. In , FtsEX is required to separate daughter cells after cell division and for viability in low-osmolarity media. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for amidase activation, but the process itself is poorly understood. Here we present the 2.1 Å structure of the FtsX periplasmic domain in complex with its periplasmic partner, EnvC. The EnvC-FtsX periplasmic domain complex has a 1-to-2 stoichiometry with two distinct FtsX-binding sites located within an antiparallel coiled coil domain of EnvC. Residues involved in amidase activation map to a previously identified groove in the EnvC LytM domain that is here found to be occluded by a "restraining arm" suggesting a self-inhibition mechanism. Mutational analysis, combined with bacterial two-hybrid screens and in vivo functional assays, verifies the FtsEX residues required for EnvC binding and experimentally test a proposed mechanism for amidase activation. We also define a predicted link between FtsEX and integrity of the outer membrane. Both the ATPase activity of FtsEX and its periplasmic interaction with EnvC are required for resistance to membrane-attacking antibiotics and detergents to which would usually be considered intrinsically resistant. These structural and functional data provide compelling mechanistic insight into FtsEX-mediated regulation of EnvC and its downstream control of periplasmic peptidoglycan amidases.
FtsEX 是一种细菌 ABC 转运蛋白，通过与质膜水解酶激活剂 EnvC 的相互作用，调节周质肽聚糖 amidase 的活性。在 ，FtsEX 是细胞分裂后分离子细胞所必需的，并且在低渗透压介质中存活所必需的。FtsEX 的 ATPase 活性及其与 EnvC 的周质相互作用对于 amidase 激活都是必需的，但该过程本身知之甚少。在这里，我们展示了 FtsX 周质结构域与周质伴侣 EnvC 形成复合物的 2.1 Å 结构。EnvC-FtsX 周质结构域复合物具有 1:2 的化学计量比，两个不同的 FtsX 结合位点位于 EnvC 的反平行卷曲螺旋结构域内。参与 amidase 激活的残基映射到 EnvC LytM 结构域中先前确定的一个沟槽上，该沟槽在此处发现被“约束臂”阻塞，表明存在自我抑制机制。突变分析，结合细菌双杂交筛选和体内功能测定，验证了 FtsEX 结合 EnvC 所必需的残基，并通过实验测试了 amidase 激活的拟议机制。我们还定义了 FtsEX 与外膜完整性之间的预测联系。FtsEX 的 ATPase 活性及其与 EnvC 的周质相互作用对于抵抗通常被认为固有抗性的膜攻击抗生素和去污剂都是必需的。这些结构和功能数据为 FtsEX 介导的 EnvC 调节及其对周质肽聚糖 amidase 的下游控制提供了令人信服的机制见解。
