DETECTION OF A CONSERVED ALPHA-HELIX IN THE KINASE-DOCKING REGION OF THE ASPARTATE RECEPTOR BY CYSTEINE AND DISULFIDE SCANNING

The transmembrane aspartate receptor of Escherichia coil and Salmonell a typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Differen t signaling states of the cytoplasmic domain modulate the kinase autop hosphorylation rate over at least a 100-fold range. Biochemical and ge netic studies have implicated a specific region of the cytoplasmic dom ain, termed the signaling subdomain, as the region that transmits regu lation from the receptor to the kinase. Here cysteine and disulfide sc anning are applied to the N-terminal half of the signaling subdomain t o probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhi bit the characteristic periodicity of an alpha-helix with distinct sol vent-exposed and buried faces. This helix, termed alpha 7, ranges appr oximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix alpha 7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Di sulfide scanning of the region suggests that helix alpha 7 is not in d irect contact with its symmetric partner (alpha 7') from the other sub unit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach term ed ''protein interactions by cysteine modification'' indicates that th e exposed C-terminal face of helix alpha 7 provides an essential docki ng site for the kinase CheA or for the coupling protein CheW.