Protein Domains and Residues Involved in the CheZ/CheAS Interaction

CheZ localizes to chemoreceptor patches by binding CheA-short (CheA_S). Residues 70 to 134 of CheZ, constituting the apical loops and part of the dimerization domain, suffice for localization. Replacements of Tyr-118, Ile-119, Leu-123, Arg-124, and Leu-126 of CheA interfere with localization. These residues are exposed in the ′P1 domain of CheA_S.CheZ is the phosphorylated CheY (CheY-P) phosphatase of Escherichia coli (5) and a number of other gram-negative bacteria (2, 3). E. coli CheZ forms a homodimer that binds two molecules of CheY-P (Fig. ​(Fig.1A)1A) (20). Within each subunit, there is an N-terminal helix of about 30 residues that is involved in negative control of phosphatase activity (16). As CheZ is depicted in Fig. ​Fig.1,1, following this helix are a sharp turn, an extended ascending helix, a hairpin loop, and an extended descending helix. The Gln-147 residues at the active sites are in the middle of the extended helical region. Following the descending helix, there is an unstructured flexible connector to the short C-terminal helix that constitutes a CheY-P binding site.Open in a separate windowFIG. 1.Regions of CheZ and CheA_S required for localization of CheZ to receptor patches. (A) A backbone trace of CheZ from the CheZ-CheY cocrystal (20) is shown. Residues Try-97 and Phe-98, mutations of which specifically inhibit CheZ-GFP localization, are shown in blue. The portion of CheZ shown in red is not sufficient to localize CheZ-GFP fusions to receptor patches. The addition of the portion of CheZ shown in green enables the CheZ-GFP fusion protein to localize to receptor patches. The disconnected helices represent the peptides that bind CheY-P and present it to the active site. (B) Backbone trace of the Salmonella CheA P1 crystal structure (12). The images shown were created from the coordinates provided in the Protein Data Bank entry for identification number 1I5N by using RasTop. The part of the P1 domain retained in ′P1 is shown in cyan. The residue at the site of phosphorylation, His-48, is shown in green. Ala or Ser substitutions for Leu-123 and Leu-126 (shown in red) strongly inhibit CheZ-GFP localization, whereas an Ala or Ser substitution for Ile-119 (shown in yellow) and the Ser substitution for Phe-118 (also yellow) moderately affect localization.Shortly above the active sites, the two hairpin loops splay out from each other. The Trp-97 and Phe-98 residues that are important for localization but not for dimerization or activity (3, 19) are located near the apical loops of CheZ. Ser substitutions for hydrophobic residues Trp-94 through Val-121 block localization but also interfere with chemotaxis (3), perhaps because they interfere with dimer formation.A short form of the CheA kinase (CheA_S) (7) begins with Met-98 of the long form of CheA (CheA_L). CheA_S is produced in a 1:2 ratio relative to CheA_L (17) and is required for the localization of CheZ to the receptor patch. CheA_S is not essential for normal chemotaxis, as assessed in standard laboratory assays (15). However, both theoretical calculations (9, 10) and in vivo measurements (19) indicate that cells that fail to localize CheZ have very different intracellular distributions of CheY-P than wild-type cells. CheZ-localizing strains show a very abrupt decline in the CheY-P concentration near the receptor patch and rather uniform levels throughout the rest of the cell. Cells in which CheZ does not localize show a gradual decline in the CheY-P concentration with distance from the receptor patch, so that CheY-P concentrations vary throughout the cell.