CheX in the Three-Phosphatase System of Bacterial Chemotaxis

Muff, Travis J.; Foster, Richard M.; Liu, Peter J. Y.; Ordal, George

doi:10.1128/jb.00896-07

Cited by 24 publications

(23 citation statements)

References 41 publications

(51 reference statements)

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“…CheA is included in the assay because of the instability of CheY-P, which requires its in situ production (11,14,42,43). FliY does not affect CheA autophosphorylation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The supernatant was used for SDS-PAGE analysis. To demonstrate the binding of various constructs of FliY (100 M) to CheY (75 M) and CheY-P (75 M), pulldown assays were performed with His-tagged proteins as described previously (42,43) with minor modifications. The samples that required phosphorylation of CheY, 20 mM acetyl phosphate (Sigma-Aldrich) in the presence of 20 mM MgCl 2 ⅐6H 2 O was added for incubation and wash steps to the binding buffer to ensure complete phosphorylation of CheY.…”

Section: Methodsmentioning

confidence: 99%

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Structure and Activity of the Flagellar Rotor Protein FliY

Sircar

Greenswag

Bilwes

et al. 2013

Journal of Biological Chemistry

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“…CheA is included in the assay because of the instability of CheY-P, which requires its in situ production (11,14,42,43). FliY does not affect CheA autophosphorylation (Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structure and Activity of the Flagellar Rotor Protein FliY

Sircar

Greenswag

Bilwes

et al. 2013

Journal of Biological Chemistry

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“…CheD binds to both the receptor and CheC in order to play a role in adaptation (23,24,32). Other work has suggested that CheD protein levels in the cell may be linked to the presence of CheC and perhaps other proteins (31).…”

Section: Quantitative Immunoblots Of the Soluble Signaling Proteinsmentioning

confidence: 99%

Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

et al. 2011

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The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B. subtilis. Significantly higher levels of chemoreceptors and much lower levels of CheA kinase were measured in B. subtilis than in Escherichia coli. The resulting cellular ratio of chemoreceptor dimers per CheA dimer in B. subtilis is roughly 23.0 ؎ 4.5 compared to 3.4 ؎ 0.8 receptor dimers per CheA dimer observed in E. coli, but the ratios of the coupling protein CheW to the CheA dimer are nearly identical in the two organisms. The ratios of CheB to CheR in B. subtilis are also very similar, although the overall levels of modification enzymes are higher. When the potential binding partners of CheD are deleted, the levels of CheD drop significantly. This finding suggests that B. subtilis selectively degrades excess chemotaxis proteins to maintain optimum ratios. Finally, the two cytoplasmic receptors were observed to localize among the other receptors at the cell poles and appear to participate in the chemoreceptor complex. These results suggest that there are many novel features of B. subtilis chemotaxis compared with the mechanism in E. coli, but they are built on a common core.Motile organisms, such as the soil-based bacterium Bacillus subtilis, have the ability to sense their chemical environment and move to more favorable conditions through a process known as chemotaxis. The chemotactic pathway in B. subtilis involves 10 different chemoreceptors and eight soluble proteins (for a review, see reference 43). The core of the chemotaxis system contains the histidine kinase CheA and the coupling protein CheW (7, 15). These proteins interact with the typically membrane-bound chemoreceptors, or MCPs (methyl-accepting chemotaxis proteins), which can sense various ligands in the extracellular environment (16,33). Once attractant binds to the receptor, CheA autophosphorylates and then transfers its phosphoryl group to CheY, the response regulator (2, 8). Phosphorylated CheY (CheYp) binds to the flagellar motor and changes the direction of its rotation, which leads to a change in the swimming behavior of the bacterium.B. subtilis employs three adaptation systems to sense chemical gradients (35). The methylation system consists of CheR, a methyltransferase, and CheB, a methylesterase, enzymes that, respectively, add and remove methyl groups at specific glutamate residues on the MCPs (4, 12, 13). A second adaptation system, the CheC-CheD-CheY system, consists of three proteins. CheC has been shown to weakly dephosphorylate CheYp, and its activity is enhanced in the presence of CheD (32,38,42). CheD, apart from interacting with CheC, can also interact with the chemoreceptors and deamidate specific glutamine residues on the MCPs, me...

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“…For each motif, the conserved glutamate and asparagine are one helical turn apart on a solvent exposed alpha helix. Mutagenesis studies (15,17,18) suggest that the (E-X 2 -N) motif mediates catalysis. The (E-X 2 -N) motifs in CheC and CheX appear comparable to the structurally unrelated CheZ phosphatase, whose active site acid and amide residues Asp143 and Gln147 are also separated by a single helical turn (9).…”

mentioning

confidence: 99%

Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate

Pazy

Motaleb

Guarnieri

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Two-component signal transduction systems are widespread in prokaryotes and control numerous cellular processes. Extensive investigation of sensor kinase and response regulator proteins from many two-component systems has established conserved sequence, structural, and mechanistic features within each family. In contrast, the phosphatases which catalyze hydrolysis of the response regulator phosphoryl group to terminate signal transduction are poorly understood. Here we present structural and functional characterization of a representative of the CheC/CheX/FliY phosphatase family. The X-ray crystal structure of Borrelia burgdorferi CheX complexed with its CheY3 substrate and the phosphoryl analogue reveals a binding orientation between a response regulator and an auxiliary protein different from that shared by every previously characterized example. The surface of CheY3 containing the phosphoryl group interacts directly with a long helix of CheX which bears the conserved (E - X2 - N) motif. Conserved CheX residues Glu96 and Asn99, separated by a single helical turn, insert into the CheY3 active site. Structural and functional data indicate that CheX Asn99 and CheY3 Thr81 orient a water molecule for hydrolytic attack. The catalytic residues of the CheX·CheY3 complex are virtually superimposable on those of the Escherichia coli CheZ phosphatase complexed with CheY, even though the active site helices of CheX and CheZ are oriented nearly perpendicular to one other. Thus, evolution has found two structural solutions to achieve the same catalytic mechanism through different helical spacing and side chain lengths of the conserved acid/amide residues in CheX and CheZ.

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CheX in the Three-Phosphatase System of Bacterial Chemotaxis

Cited by 24 publications

References 41 publications

Structure and Activity of the Flagellar Rotor Protein FliY

Structure and Activity of the Flagellar Rotor Protein FliY

Cellular Stoichiometry of the Chemotaxis Proteins in Bacillus subtilis

Identical phosphatase mechanisms achieved through distinct modes of binding phosphoprotein substrate

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