Phospho-mimetic CheV interacts with a subset of chemoreceptors

Chemotaxis pathways are among the most complex signaling systems in bacteria. A central feature of these pathways is the ternary complex formed by chemoreceptors, the autokinase CheA, and the coupling proteins CheW and CheV. Whereas CheW is present in all chemotaxis pathways, CheV is primarily found in bacteria that contain many chemoreceptors. CheV is a fusion of a CheW-like domain to a phosphorylatable receiver domain. The roles of CheV and its phosphorylation are currently uncertain. Pseudomonas aeruginosa contains many chemoreceptors for which the cognate signals have been identified. Quantitative capillary chemotaxis assays of a cheV mutant revealed that responses to certain chemoeffectors, such as nitrate and α-ketoglutarate, were drastically reduced, while responses to others, such as amino acids and inorganic phosphate, were comparable to the wild type, indicating that CheV selectively acts on specific chemoreceptors. To study the mechanism of CheV action, we conducted protein-protein interaction experiments using isothermal titration calorimetry. These studies showed that unphosphorylated CheV fails to bind to cytosolic fragments of the McpN and PctA chemoreceptors, which mediate responses to nitrate and amino acids, respectively. In contrast, the phosphorylation-mimic CheV D238E bound with very high affinity (K_D = 8 nM) to McpN but failed to interact with PctA. Thus, CheV in P. aeruginosa binds to some chemoreceptors but not to others in a phosphorylation-dependent manner. These results suggest that CheV is a regulatory protein that modulates signaling through specific chemoreceptors. CheV may thus facilitate the coordination of chemotaxis responses in complex, multi-chemoreceptor systems.IMPORTANCECheV is one of the least understood chemosensory signaling proteins. Our demonstration that CheV interacts only with certain chemoreceptors offers fundamental new insights. These findings, combined with the observation that CheV is present in bacteria with numerous chemoreceptors, suggest that CheV plays a role in coordinating chemotactic outputs in complex chemosensory systems. Understanding the mechanisms by which chemotactic responses are defined in bacteria with a high number of chemoreceptors is a major research priority in the field of chemotaxis. While previous studies, including this one, show that the ability to be phosphorylated is crucial for CheV function, the molecular consequences of CheV phosphorylation have remained unclear. Our discovery that phosphorylation is essential for CheV binding to certain chemoreceptors fills in this critical gap in understanding the molecular mechanism of CheV. This study is likely to inspire further research into CheV function in other bacteria using similar approaches.