Thanikkal Edvin J, Mangu Jagadish C K, Francis Matthew S
Department of Molecular Biology, Umeå University, Umeå, SE-901 87, Sweden.
BMC Res Notes. 2012 Sep 27;5:536. doi: 10.1186/1756-0500-5-536.
The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.
CpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the λcI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.
This interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.
CpxA传感器激酶-CpxR反应调节因子双组分调节系统是细菌包膜完整性的哨兵。它整合多种信号，可改变大量组分的表达，这些组分有助于保护包膜免受损伤并促进细菌存活。在诸如假结核耶尔森菌等细菌病原体中，这也延伸至发病机制。CpxR被认为在传感器激酶CpxA磷酸化后会形成二聚体。这种磷酸化使CpxR能够结合特定的DNA序列，在那里它作用于基因转录。由于Cpx途径的激活依赖于蛋白质-蛋白质相互作用，我们对假结核耶尔森菌的CpxR和CpxA进行了相互作用分析。
对包含或缺乏假定内部连接子的CpxR全长和截短版本进行了评估，以确定它们同源二聚化以及与CpxA相互作用的能力。使用基于腺苷酸环化酶的细菌双杂交方法，全长CpxR很容易与CpxA结合。CpxR的N末端也能与自身以及全长CpxR同源二聚化。基于λcI阻遏物的第二种同源二聚化测定也表明CpxR的C末端可以同源二聚化。虽然连接子不是特别必需的，但它增强了CpxR的同源二聚化。对cpxR进行诱变确定了第51位残基处的天冬氨酸、假定的N末端卷曲螺旋和C末端翼状螺旋-转角-螺旋结构域是CpxR同源二聚化的介导因子。对CpxA全长和截短版本的仔细研究表明，二聚化涉及N末端以及一个内部二聚化和组氨酸磷酸转移结构域。
这种相互作用分析确定了CpxR和CpxA中负责自身相互作用或彼此相互作用的区域。当与其他生理和生化测试相结合时，这两种基于杂交的测定方法都有助于剖析可能是Cpx途径激活和抑制基础的分子接触。
