Mutational Analysis of the Chemoreceptor-Coupling Domain of the Escherichia coli Chemotaxis Signaling Kinase CheA

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/P3 interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA. The histidine kinase CheA plays a central signaling role in bacterial chemotaxis pathways (see references 48 and 49 for recent reviews). In Escherichia coli, CheA forms signaling complexes with five membrane-associated chemoreceptors, known as methyl-accepting chemotaxis proteins (MCPs), that communicate with the flagellar motors by controlling CheA activity. CheA autophosphorylates at a histidine residue, using ATP as the phosphodonor, and subsequently donates those phosphoryl groups to two response regulators, CheY and CheB. Phosphorylated CheY (phospho-CheY) interacts with the switching machinery at the base of the flagellar motors to promote clockwise (CW) rotation, which causes random turning episodes or tumbles during swimming. Phospho-CheB functions in a feedback adaptation circuit that enables the cells to detect temporal changes in attractant and repellent concentrations as they move through spatial chemical gradients. Chemoeffectors, sensed by the chemoreceptors, modulate CheA activity over a wide range. Unliganded receptors activate CheA several hundredfold over its basal, receptor-uncoupled autophosphorylation rate, whereas attractant-bound receptors deactivate CheA to about its basal activity level (8, 9, 26, 33, 45). CheA autophosphorylation control occurs in ternary complexes formed between the cytoplasmic signaling domains of the chemoreceptors, CheA, and the coupling protein CheW, which is essential for chemotactic behavior in vivo and for receptor-mediated activation of CheA in vitro. The role of CheW in receptor coupling control of CheA is poorly understood, owing to a paucity of structural information about the