Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution
Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution
复制标题
DOI：
10.1073/pnas.1821959116
复制
发表时间：
2019-03
影响因子：
11.1
通讯作者：
Kaiming Zhang;Huawei Zhang;Shanshan Li;G. Pintilie;T. Mou;Yuanzhu Gao;Qinfen Zhang;H. van den Bedem;M. Schmid;S. Au;W. Chiu
中科院分区：
文献类型：
--
作者：
Kaiming Zhang;Huawei Zhang;Shanshan Li;G. Pintilie;T. Mou;Yuanzhu Gao;Qinfen Zhang;H. van den Bedem;M. Schmid;S. Au;W. Chiu
幽门螺杆菌感染了近一半的世界人口，是各种胃病的主要原因。它进化出各种毒力因子来帮助其宿主定植和感染，包括负责H发病机制的空泡细胞毒素A（VacA）。幽门相关疾病在这里，我们解决多个结构的水溶性VacA低聚物组件使用冷冻电子显微镜（cryo-EM）在近原子分辨率。这些研究表明，跨膜的孔形成过程所需的功能VacA六聚体的结构变化的模型，并强调了冷冻EM的能力，以解决多个结构快照从一个单一的标本在近原子分辨率。幽门螺杆菌（Helicobacter pylori，H.幽门螺杆菌）是胃癌的主要危险因素，并且是最普遍的致癌感染因子之一。溶血性细胞毒素A（VacA）是H. pylori并诱导多种细胞反应。虽然VacA的结构和功能研究已被广泛进行，高分辨率的全长VacA原聚体的结构和寡聚化的分子基础仍然是未知的。在这里，我们使用冷冻电子显微镜来解决10个结构的VacA组件，包括单层（六聚体和七聚体）和双层（十二聚体，十三聚体和十四聚体）低聚物。从近原子分辨率图谱中得到的88-kDa全长VacA原聚体的模型在不同寡聚体中高度保守，并且显示出由具有广泛结构域-结构域相互作用的两个结构域组成的连续右手β-螺旋。在同一层中稳定低聚物的相邻原聚体之间的特定相互作用被很好地解析。对于双层低聚物，我们发现短和/或长距离的疏水相互作用的两层之间的原聚体。我们的结构和其他以前的观察导致一个机制模型，其中VacA六聚体将对应于prepore-forming状态，和N-末端区域的VacA负责膜插入将经历一个大的构象变化，使疏水跨膜区域的中心的寡聚体的膜通道的形成。
Significance Helicobacter pylori infects nearly half of the world’s population and is the primary cause of various gastric diseases. It has evolved various virulence factors to aid its host colonization and infection, including the vacuolating cytotoxin A (VacA) that is responsible for the pathogenesis of H. pylori-related diseases. Here, we resolve multiple structures of the water-soluble VacA oligomeric assemblies using cryoelectron microscopy (cryo-EM) at near-atomic resolution. These studies suggest a model of structural changes of functional VacA hexamer needed for the pore-formation process across the membrane and highlight the capability of cryo-EM to resolve multiple structure snapshots from a single specimen at near-atomic resolution. Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed β-helix made up of two domains with extensive domain–domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.