Roehrich A Dorothea, Bordignon Enrica, Mode Selma, Shen Da-Kang, Liu Xia, Pain Maria, Murillo Isabel, Martinez-Argudo Isabel, Sessions Richard B, Blocker Ariel J
From the School of Cellular and Molecular Medicine and School of Biochemistry, Medical Sciences Building, Faculty of Medical and Veterinary Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
the Fachbereich Physik, Freie Universität Berlin, 14195 Berlin, Germany.
J Biol Chem. 2017 Feb 3;292(5):1705-1723. doi: 10.1074/jbc.M116.746826. Epub 2016 Dec 14.
Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in Shigella As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.
III型分泌系统是一种复杂的纳米机器，用于将革兰氏阴性菌中的蛋白质注入真核细胞。尽管它们在环境条件适宜时组装，但只有在与宿主细胞接触时才开始分泌。分泌是分层进行的。首先，形成孔道的转运蛋白被释放。其次，效应蛋白被注入。这些蛋白类别之间的层级关系由志贺氏菌中一种保守的守门蛋白MxiC介导。由于其分子作用机制仍知之甚少，我们利用其结构指导定点诱变并剖析其功能。我们鉴定出主要影响MxiC调控所有已知特征的突变体如下：转运蛋白、MxiC和/或效应蛋白的分泌。然后，我们利用分子遗传学确定这些突变体在调控级联反应中的受影响点。对其中一些突变体的分析促使我们进行了一系列电子顺磁共振实验，这些实验提供了证据表明MxiC直接与IpaD相互作用。我们提出了这种相互作用如何调节其构象转换的方式，而这种构象转换对其功能至关重要。
