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 coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase. Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an alpha-helix with distinct solvent-exposed and buried faces. This helix, termed alpha7, ranges approximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix alpha7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix alpha7 is not in direct contact with its symmetric partner (alpha7') from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix alpha7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.
Bass
R B
RB
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, USA.
Falke
J J
JJ
D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002633:Chemotaxis; D003545:Cysteine; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017459:Receptors, Amino Acid / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D017459:Receptors, Amino Acid; D011994:Recombinant Proteins; C031959:aspartic acid receptor; C051236:CheW protein, Bacteria; D001224:Aspartic Acid; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D003545:Cysteine