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首页> 外文期刊>Journal of bacteriology >Functional Determinants of a Small Protein Controlling a Broadly Conserved Bacterial Sensor Kinase
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Functional Determinants of a Small Protein Controlling a Broadly Conserved Bacterial Sensor Kinase

机译：控制宽保守的细菌传感器激酶的小蛋白的功能确定剂

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The PhoQ/PhoP two-component system plays a vital role in the regulation of Mg~(2+) homeostasis, resistance to acid and hyperosmotic stress, cationic antimicrobial peptides, and virulence in Escherichia coli, Salmonella , and related bacteria. Previous studies have shown that MgrB, a 47-amino-acid membrane protein that is part of the PhoQ/PhoP regulon, inhibits the histidine kinase PhoQ. MgrB is part of a negative-feedback loop modulating this two-component system that prevents hyperactivation of PhoQ and may also provide an entry point for additional input signals for the PhoQ/PhoP pathway. To explore the mechanism of action of MgrB, we analyzed the effects of point mutations, C-terminal truncations, and transmembrane (TM) region swaps on MgrB activity. In contrast to two other known membrane protein regulators of histidine kinases in E. coli, we found that the MgrB TM region is necessary for PhoQ inhibition. Our results indicate that the TM region mediates interactions with PhoQ and that W20 is a key residue for PhoQ/MgrB complex formation. Additionally, mutations of the MgrB cytosolic region suggest that the two N-terminal lysines play an important role in regulating PhoQ activity. Alanine-scanning mutagenesis of the periplasmic region of MgrB further indicated that, with the exception of a few highly conserved residues, most residues are not essential for MgrB’s function as a PhoQ inhibitor. Our results indicate that the regulatory function of the small protein MgrB depends on distinct contributions from multiple residues spread across the protein. Interestingly, the TM region also appears to interact with other noncognate histidine kinases in a bacterial two-hybrid assay, suggesting a potential route for evolving new small-protein modulators of histidine kinases. IMPORTANCE One of the primary means by which bacteria adapt to their environment is through pairs of proteins consisting of a sensor and a response regulator. A small membrane protein, MgrB, impedes the activity of sensor protein PhoQ, thereby affecting the expression of PhoQ regulated virulence genes in pathogenic bacteria. However, it is unknown how such a small protein modulates the activity of PhoQ. Here, we studied the functional determinants of MgrB and identified specific amino acids critical for the protein's inhibitory function. Notably, we find that the membrane-spanning region is important for MgrB interaction with PhoQ. Additionally, this region appears to physically interact with other sensors, a property that may be important for evolving small protein regulators of sensor kinases.
机译：Phoq / Phop双组分系统在Mg〜（2+）稳态的调节中起到重要作用，对大肠杆菌，沙门氏菌和相关细菌的酸和高血压胁迫，阳离子抗菌药物，阳离子抗菌肽和毒力。先前的研究表明，MGRB，一种是Phoq / Phop调节件的一部分的47-氨基酸膜蛋白，抑制组氨酸激酶phoq。 MGRB是负反馈回路的一部分，调制防止PHOQ的多动激活的双组分系统，并且还可以为PHOQ / PHOP路径提供额外输入信号的入口点。为了探讨MGRB的作用机制，我们分析了点突变，C末端截短和跨膜（TM）区交换对MGRB活性的影响。与大肠杆菌中的另外两种其他已知的膜蛋白调节剂相反，我们发现MGRB TM区域是PHOQ抑制所必需的。我们的结果表明，TM区介导与PHOQ的相互作用，并且W20是PHOQ / MGRB复合物形成的关键残留物。另外，MGRB细胞源区的突变表明，两种N-末端赖氨酸在调节PHOQ活性方面发挥着重要作用。 MGRB的周质区的丙氨酸扫描诱变进一步表明，除了几种高度保守的残基外，大多数残基对MGRB的功能作为PhoQ抑制剂至关重要。我们的结果表明，小蛋白质MGRB的调节功能取决于来自蛋白质的多个残留物的不同贡献。有趣的是，TM区域还似乎在细菌双杂交测定中与其他非同一性组氨酸激酶相互作用，表明用于演化组氨酸激酶的新的小蛋白质调节剂的潜在途径。细菌适应环境的主要方法之一是通过由传感器和响应调节器组成的蛋白质对。小膜蛋白，MGRB，阻碍了传感器蛋白PHOQ的活性，从而影响致病细菌中PHOQ受毒力基因的表达。然而，尚不清楚这种小蛋白质如何调节Phoq的活性。在这里，我们研究了MGRB的功能决定因素，并确定了对蛋白质的抑制作用致力于关键的特异性氨基酸。值得注意的是，我们发现膜跨越区域对于与Phoq的MGRB相互作用很重要。另外，该区域似乎与其他传感器物理地相互作用，这对于改进传感器激酶的小蛋白调节剂可能是重要的一种性质。

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