New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli
New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli
复制标题
DOI：
10.1074/jbc.m110.196923
复制
发表时间：
2011-03-25
影响因子：
4.8
通讯作者：
Jung, Kirsten
中科院分区：
文献类型：
--
作者：
Haneburger, Ina;Eichinger, Andreas;Jung, Kirsten
大肠埃希菌的膜整合转录调控因子CADC在低的外部pH条件下激活了CadBA操纵子的表达，并伴随着有效的赖氨酸，提供了对弱酸性胁迫的适应。CADC是ToxR类蛋白的代表，它在单个多肽中结合了感觉、信号转导和DNA结合活性。尽管CADC等几种ToxR类调控因子以及霍乱弧菌毒力的主要调节因子ToxR本身在酸性pH条件下激活基因表达已被广泛研究，但其分子激活机制尚不清楚。在这项研究中，结构导向的突变分析被用来阐明CADC检测外部环境酸化的机制。因此，一簇带负电荷的氨基酸(Asp-198、Asp-200、Glu-461、Glu-468和Asp-471)被发现对pH检测至关重要。这些氨基酸在CADC的周质结构域表面形成一个带负电荷的斑块，横跨其两个亚结构域。该补丁内氨基酸替换的不同组合的结果表明，N-末端亚区整合并将来自这两个亚区的信号传递到跨膜区。磷脂组成的变化不会影响依赖于pH的钙结合蛋白的表达，因此，酸性表面斑块与带负电荷的头基不太可能相互作用。根据这些模型，这些酸性氨基酸侧链的质子化减少了CADC二聚体中两个亚区之间和/或两个单体之间的排斥力，从而在环境pH降低时能够激活受体。
The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.