Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ

Zhao, Rui; Collins, Edward J.; Bourret, Robert B.; Silversmith, Ruth E.

doi:10.1038/nsb816

Cited by 118 publications

(197 citation statements)

References 40 publications

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“…These proteins might still act in a similar manner. Based on the x-ray diffraction structure for CheZ in complex with CheY, it is believed that glutamine residue 147 of CheZ contributes in the release of phosphate from CheY-P by positioning and activating a water molecule in the CheY-P active site (35). Although no glutamine residues are conserved in CheC and FliY, other highly conserved residues that are possibly capable of exercising a similar function include an aspartate, a serine, two glutamates and two asparagines (Fig.…”

Section: Figmentioning

confidence: 99%

Bacillus subtilis CheC and FliY Are Members of a Novel Class of CheY-P-hydrolyzing Proteins in the Chemotactic Signal Transduction Cascade

Szurmant¹,

Muff²,

Ordal

2004

Journal of Biological Chemistry

115

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Rapid restoration of prestimulus levels of the chemotactic response regulator, CheY-P, is important for preparing bacteria and archaea to respond sensitively to new stimuli. In an extension of previous work (Szurmant, H., Bunn, M. W., Cannistraro, V. J., and Ordal, G. W. (2003) J. Biol. Chem. 278, 48611-48616), we describe a new family of CheY-P phosphatases, the CYX family, that is widespread among the bacteria and archaea. These proteins provide another pathway, in addition to the ones involving CheZ of the ␥-and ␤-proteobacteria (e.g. Escherichia coli) or the alternative CheY that serves as a "phosphate sink" among the ␣-proteobacteria (e.g. Sinorhizobium meliloti), for dephosphorylating CheY-P. In particular, we identify CheC, known previously to be involved in adaptation to stimuli in Bacillus subtilis, as a CheY-P phosphatase. Using an in vitro assay used previously to demonstrate that the switch protein FliY is a CheY-P phosphatase, we have shown that increasing amounts of CheC accelerate the hydrolysis of CheY-P. In vivo, a double mutant lacking cheC and the region of fliY that encodes the CheY-P binding domain is almost completely smooth swimming, implying that these cells contain very high levels of CheY-P. CheC appears to be primarily involved in restoring normal CheY-P levels following the addition of attractant, whereas FliY seems to act on CheY-P constitutively. The activity of CheC is relatively low compared to that of FliY, but we have shown that the chemotaxis protein CheD enhances the activity of CheC 5-fold. We suggest a model for how FliY, CheC, and CheD work together to regulate CheY-P levels in the bacterium.

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Section: Figmentioning

confidence: 99%

Bacillus subtilis CheC and FliY Are Members of a Novel Class of CheY-P-hydrolyzing Proteins in the Chemotactic Signal Transduction Cascade

Szurmant¹,

Muff²,

Ordal

2004

Journal of Biological Chemistry

115

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“…It is not surprising therefore that many RRs utilize the α4-β5-α5 face for regulatory protein-protein interactions, the affinities of which are altered by phosphorylation. These interactions form the basis for a variety of different inter-and intramolecular regulatory mechanisms such as the binding of CheY-P to FliM 59 and CheZ, 60 the formation of homodimers of phosphorylated FixJ, 46 the alternation between different dimeric states in DctD, 61,62 and the inhibitory contacts between the unphosphorylated regulatory and effector domains of NarL 27 and CheB. 28 An analysis of residue conservation at the α4-β5-α5 face of the three major subfamilies of RR transcription factors (OmpR/PhoB, NarL/FixJ, and NtrC/DctD) revealed important differences that distinguish the OmpR/PhoB subfamily from the others.…”

Section: The α4-β5-α5 Facementioning

confidence: 99%

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

Toro-Roman

Mack²,

Stock

2005

Journal of Molecular Biology

119

137

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SUMMARYEscherichia coli react to changes from aerobic to anaerobic conditions of growth using the ArcAArcB two-component signal transduction system. This system, in conjunction with other proteins, regulates the respiratory metabolic pathways in the organism. ArcA is a member of the OmpR/ PhoB subfamily of response regulator transcription factors that are known to regulate transcription by binding in tandem to target DNA direct repeats. It is still unclear in this subfamily how activation by phosphorylation of the regulatory domain of response regulators stimulates DNA binding by the effector domain and how dimerization and domain orientation, as well as intra-and intermolecular interactions, affect this process. In order to address these questions we have solved the crystal structure of the regulatory domain of ArcA in the presence and absence of the phosphoryl analog, BeF 3 − . In the crystal structures, the regulatory domain of ArcA forms a symmetric dimer mediated by the α4-β5-α5 face of the protein and involving a number of residues that are highly conserved in the OmpR/PhoB subfamily. It is hypothesized that members of this subfamily use a common mechanism of regulation by dimerization. Additional biophysical studies were employed to probe the oligomerization state of ArcA, as well as its individual domains, in solution. The solution studies show the propensity of the individual domains to associate into oligomers larger than the dimer observed for the intact protein, and suggest that the C-terminal DNA-binding domain also plays a role in oligomerization.

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“…This results in an increase in the CCW flagellar rotation. Zhao et al, 2002). CheD deamidates certain selected glutamine residues in the receptors, changing them to glutamate residues (Kristich & Ordal, 2002), a reaction that makes the receptors more amenable to stimulation by attractant, possibly due to an effect on higher-order complexes within receptor clusters (Kirby et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis

2004

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Bacillus subtilis has a more complex mechanism of chemotaxis than does the paradigm organism, Escherichia coli. In order to understand better the role of the novel chemotaxis proteins -CheC, CheD and CheV -mutants in which increasing numbers of the corresponding genes had been deleted were studied as tethered cells and their biases and sometimes durations of counterclockwise (CCW) and clockwise (CW) flagellar rotations in response to addition and removal of the attractant asparagine were observed. The cheC mutant was found to have considerably reduced switching frequency (that is, prolonged CCW and CW rotations) without a significantly different prestimulus CCW bias, compared with wild-type. This result may indicate that in absence of CheC the switch might be in a conformation less resembling the transition state than in presence of CheC. Conversely, the cheB (methylesterase) mutant showed considerably increased switching frequency without affecting CCW bias, compared with wild-type. Removal of all known adaptation systems -the methylation, CheC and CheV systems -resulted in a mutant (cheRBCDV ) that still retained some adaptation following the addition of attractant.

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Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ

Cited by 118 publications

References 40 publications

Bacillus subtilis CheC and FliY Are Members of a Novel Class of CheY-P-hydrolyzing Proteins in the Chemotactic Signal Transduction Cascade

Bacillus subtilis CheC and FliY Are Members of a Novel Class of CheY-P-hydrolyzing Proteins in the Chemotactic Signal Transduction Cascade

Structural Analysis and Solution Studies of the Activated Regulatory Domain of the Response Regulator ArcA: A Symmetric Dimer Mediated by the α4-β5-α5 Face

Effect of loss of CheC and other adaptational proteins on chemotactic behaviour in Bacillus subtilis

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