Kotov Vadim, Lunelli Michele, Wald Jiri, Kolbe Michael, Marlovits Thomas C
Centre for Structural Systems Biology (CSSB), Notkestraße 85, 22607 Hamburg, Germany.
University Medical Centre Hamburg-Eppendorf (UKE), Martinistraße 52, 20246 Hamburg, Germany.
Biochem Biophys Rep. 2021 Jun 27;27:101039. doi: 10.1016/j.bbrep.2021.101039. eCollection 2021 Sep.
Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.
革兰氏阴性病原体进化出一种类似注射器的纳米机器，称为III型分泌系统，用于将蛋白质效应物输送到宿主细胞的细胞质中。该系统的一个重要组成部分是一根长长的螺旋状针状细丝，它从细菌表面伸出，连接细菌和真核细胞的细胞质。以前的结构研究主要集中在重组的III型针状细丝上，而这些细丝缺乏生物学背景。在这项工作中，我们介绍了一种简便的方法，以获得附着在III型分泌系统基体上的针状细丝的高分辨率冷冻电镜结构。我们通过解析PrgI细丝的结构验证了我们的方法，并通过获得志贺氏菌MxiH细丝的首个高分辨率冷冻电镜重建来证明其效用。我们的工作为在诱变研究、蛋白质结构进化和药物开发的背景下对附着的III型针状细丝进行系统的结构表征铺平了道路。
