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首页> 外文期刊>Journal of Molecular Biology >The Cytoplasm-Entry Domain of Antibacterial CdiA Is a Dynamic alpha-Helical Bundle with Disulfide-Dependent Structural Features
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The Cytoplasm-Entry Domain of Antibacterial CdiA Is a Dynamic alpha-Helical Bundle with Disulfide-Dependent Structural Features

机译：抗菌Cdia的细胞质入口结构域是一种具有二硫化物依赖性结构特征的动态α-螺旋束

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Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to compete with neighboring cells. CDI+ strains express cell-surface CdiA effector proteins, which carry a toxic C-terminal region (CdiA-CT) that is cleaved from the effector upon transfer into the periplasm of target bacteria. The released CdiA-CT consists of two domains. The C-terminal domain is typically a nuclease that inhibits cell growth, and the N-terminal "cytoplasm-entry" domain mediates toxin translocation into the target-cell cytosol. Here, we use NMR and circular dichroism spectroscopic approaches to probe the structure, stability, and dynamics of the cytoplasm-entry domain from Escherichia coli STEC_MH1813. Chemical shift analysis reveals that the CdiA-CTMHI1813 entry domain is composed of a C-terminal helical bundle and a dynamic N-terminal region containing two disulfide linkages. Disruption of the disulfides by mutagenesis or chemical reduction destabilizes secondary structure over the N-terminus, but has no effect on the C-terminal helices. Although critical for N-terminal structure, the disulfides have only modest effects on global thermodynamic stability, and the entry domain exhibits characteristics of a molten globule. We find that the disulfides form in vivo as the entry domain dwells in the periplasm of inhibitor cells prior to target-cell recognition. CdiA-CTMHI813 variants lacking either disulfide still kill target bacteria, but disruption of both bonds abrogates growth inhibition activity. We propose that the entry domain's dynamic structural features are critical for function. In its molten globule-like state, the domain resists degradation after delivery, yet remains pliable enough to unfold for membrane translocation. (C) 2019 Published by Elsevier Ltd.
机译：许多革兰阴性细菌种类使用接触依赖性生长抑制（CDI）系统与相邻细胞竞争。 CDI +菌株表达细胞表面CDIA效应蛋白，其携带毒性C末端区域（CDIA-CT），其在转移到靶细菌的周质上时从效应子中切割。释放的CDIA-CT由两个域组成。 C-末端结构域通常是抑制细胞生长的核酸酶，N-末端“细胞质 - 进入”结构域介导毒素易位进入靶细胞胞质细胞溶胶。在这里，我们使用NMR和圆形二色性光谱方法来探讨来自大肠杆菌STEC_MH1813的细胞质入口结构域的结构，稳定性和动力学。化学换档分析显示Cdia-CTMHI1813进入结构域由C末端螺旋束和含有两种二硫键键的动态N-末端区域组成。通过诱变或化学减少破坏二硫化的破坏稳定在N-末端上的二次结构，但对C末端螺旋没有影响。虽然N-末端结构至关重要，但二硫化物对全局热力学稳定性仅适度影响，并且进入结构域表现出熔融球的特征。我们发现，在靶细胞识别之前，在抑制剂细胞的周质中，在体内含有二硫化物形式。 CDIA-CTMHI813缺乏二硫化物仍然杀死靶细菌的变体，但两种键的破坏废除生长抑制活性。我们建议进入域的动态结构特征对于功能至关重要。在其熔化的球状状态下，畴抵抗递送后的降解，但对于膜易位仍然足够柔韧。 （c）2019年由elestvier有限公司发布

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