Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.
Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.
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沙门氏菌SPI-2注射体中SctV （SsaV）细胞质结构域的结构及其pH感应机制的意义
DOI：
10.1016/j.jsb.2021.107729
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发表时间：
2021-06
影响因子：
3
通讯作者：
Beeby M
中科院分区：
文献类型：
--
作者：
Matthews-Palmer TRS;Gonzalez-Rodriguez N;Calcraft T;Lagercrantz S;Zachs T;Yu XJ;Grabe GJ;Holden DW;Nans A;Rosenthal PB;Rouse SL;Beeby M
全长III型分泌系统SctV的冷冻电镜可分解细胞质而非跨膜结构域。MD仿真表明SctV原体具有柔性铰链。酸化通过改变原体间相互作用扩大SctV环。细菌III型分泌系统集合了注射体和鞭毛的轴向结构。注射型分泌系统随后通过它们的空心针将效应蛋白分泌到宿主体内，这需要协调。在肠炎沙门氏菌血清型鼠伤寒杆菌SPI-2注射体中，该开关通过感知宿主细胞质的中性pH值而触发。特异性转换的核心是具有n端跨膜结构域和环状c端细胞质结构域的非美SctV蛋白。一个“守门人”复合体以pH依赖的方式与SctV细胞质结构域相互作用，促进转座子分泌，同时通过一种尚不清楚的机制抑制效应物分泌。为了更好地了解SctV在SPI-2跨位点效应物特异性切换中的作用，我们纯化了全长SctV，并使用冷冻电镜（cryo-EM）测定了其环状细胞质区域的结构。结构比较和分子动力学模拟表明，细胞质环面是由其核心子结构域3稳定的，子结构域2和子结构域4围绕其铰链，改变了涉及守门人和底物结合的柔性外间隙。根据表面守恒、去质子化和结构运动的模式，先前确定的关键残基的位置表明，gatekeeper结合了一个埋在邻近子结构域4s之间的裂缝。模拟表明，局部pH值从5到7.2的变化稳定了亚结构域3铰链，并缩小了非美环体的中心孔径。我们的结果与SctV的局部pH感应模型一致，其中SctV细胞质结构域的pH依赖动力学影响守门人复合物的结合。
CryoEM of a full-length type III secretion system SctV resolves cytoplasmic but not transmembrane domains. MD simulations show SctV protomers flexibly hinge. Acidification expands the SctV ring by altering interprotomer interactions. Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A ‘gatekeeper’ complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region’s structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
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作者：
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DOI：
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发表时间：
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