A FRAGMENT LIBERATED FROM THE ESCHERICHIA-COLI CHEA KINASE THAT BLOCKS STIMULATORY, BUT NOT INHIBITORY, CHEMORECEPTOR SIGNALING

CheA, a cytoplasmic histidine autokinase, in conjunction with the CheW coupling protein; forms stable ternary complexes with the cytoplasmic signaling domains of transmembrane chemoreceptors. These signaling co mplexes induce chemotactic movements by stimulating or inhibiting CheA autophosphorylation activity in response to chemoeffector stimuli. To explore the mechanisms of CheA control by chemoreceptor signaling com plexes, we examined the ability of various CheA fragments to interfere with receptor coupling control of CheA. CheA[250-654], a fragment car rying the catalytic domain and an adjacent C-terminal segment previous ly implicated in stimulatory control of CheA activity, interfered with the Production of clockwise flagellar rotation and with chemotactic a bility in wild-type cells. Epistasis tests indicated that CheA[250-654 ] blocked clockwise rotation by disrupting stimulatory coupling of Che A to receptors. In vitro coupling assays confirmed that a stoichiometr ic excess df CheA[250-654] fragments could exclude CheA from stimulato ry receptor complexes, most likely by competing for CheW binding. Howe ver, CheA[250-654] fragments; even in vast excess, did not block recep tor-mediated inhibition of CheA, suggesting that CheA[250-654] lacks a n inhibitory contact site present in native CheA. This inhibitory targ et is most likely in the N-terminal P1 domain, which contains His-48, the site of autophosphorylation. These findings suggest a simple allos teric model of CheA control by ternary signaling complexes in which th e receptor signaling domain conformationally regulates the interaction between the substrate and catalytic domains of CheA.