Specific contacts between chemoreceptors and the coupling protein CheW are differentially affected during signaling

ANDREA PEDETTA; CRISTINA CHIAPPE; CLAUDIA STUDDERT

Rosario

Congreso; XIX Congreso Argentino de Microbiología General; 2013

Sociedad Argentina de Microbiología General

Bacterial chemotaxis allows microorganisms to sense their immediate chemical environment and modulate their movement accordingly. The main components of this signal transduction system are chemoreceptors, which sense the chemical stimuli and control the activity of the histidine kinase CheA, within a ternary complex that also contains the adaptor protein CheW. The activity of the kinase controls the levels of phosphorylated CheY, which communicates the sensory input to the flagellar motors. To accomplish its function, CheW directly interacts with receptors and with CheA. The domain of CheA that interacts with CheW (P5 domain) is homologous to CheW itself, and both domains show significant structure and sequence conservation. The interaction of CheW with chemoreceptors occurs through a hydrophobic surface between the two b-barrels of the protein. CheA is proposed to interact with the receptors through the corresponding region of its P5 domain. The actual changes that occur within the ternary complex to modulate kinase activity during signaling are still being investigated. Several models have been proposed from structural and biochemical studies using in vitro approaches. In order to test whether changes in the organization of the ternary complex can be detected in whole cells, we sought for specific contacts between chemoreceptors and CheW that could be targets of disulfide crosslinking. We found pairs of cysteine replacements that form disulfides upon treatment of whole cells with the oxidant diamide. Disulfide formation was significantly reduced when a mutant version with altered affinity with receptors was used, indicating that crosslinking indeed reflected a specific interaction between both proteins. Then, we characterized the efficiency of crosslinking under different conditions. Interestingly, two different crosslinking pairs showed opposite behavior upon attractant stimuli. This suggests the occurrence of a signal-dependent relative movement of CheW with respect to the receptors rather than a change in the association between the two proteins. The presence/absence of CheA in the cell also affected in opposite ways the crosslinking efficiency of these two pairs, suggesting that the P5 domain might compete with CheW more efficiently for one of the interacting points, while favoring the interaction through the other contact. The obtained results are discussed under the light of current experimental evidence.