Protein Phosphorylation and Bacterial Chemotaxis

Hess, Judith; Bourret, Robert B.; Oosawa, Kenji; Matsumura, Philip; Simon, Melvin I.

doi:10.1101/sqb.1988.053.01.008

Cited by 51 publications

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“…Thus, high intracellular concentrations of phospho-CheY correlate with the tumbling motion of the cell. Smooth swimming is restored by dephosphorylation of CheY, which may be due to both the spontaneous hydrolysis of the phosphate group and the activity of the CheZ protein, which strongly accelerates the dephosphorylation of phospho-CheY (9,11). It has also been established that phospho-CheY binds to the CheZ protein and that, following dephosphorylation, CheY is released from CheZ (4,18).…”

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confidence: 95%

“…The signal transduction that ultimately controls the frequency of switches in flagellar rotation is mediated by several transmembrane receptors and by a series of cytoplasmic proteins. In Escherichia coli and Salmonella typhimurium, the external message is transmitted through transmembrane receptors to a cytoplasmic autophosphorylating histidine kinase, CheA, which transfers its phosphoryl group to the response regulator CheY (5,9,12,26). CheY in its phosphorylated form (phospho-CheY) binds to the flagellar switch (2,17,25) and causes clockwise rotation of the flagella, resulting in a tumbling event (7,15).…”

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In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants

Sanna

1996

J Bacteriol

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Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain-or loss-of-function mutant proteins define separate functional domains of the CheZ protein.Peritrichously flagellated bacteria are able to move towards high concentrations of attractant substances by alternating the rotational direction of their flagella (for reviews, see references 1, 6, 16, and 22 to 24). Counterclockwise rotation results in unidirectional swimming (smooth swimming), whereas changes in the swimming direction (tumbling) are achieved by intermittent clockwise rotation of the flagella. Cells swimming toward increasing concentrations of attractant tend to tumble less frequently, thereby biasing their direction of movement towards regions of higher attractant concentration (3, 21). The signal transduction that ultimately controls the frequency of switches in flagellar rotation is mediated by several transmembrane receptors and by a series of cytoplasmic proteins. In Escherichia coli and Salmonella typhimurium, the external message is transmitted through transmembrane receptors to a cytoplasmic autophosphorylating histidine kinase, CheA, which transfers its phosphoryl group to the response regulator CheY (5, 9, 12, 26). CheY in its phosphorylated form (phospho-CheY) binds to the flagellar switch (2, 17, 25) and causes clockwise rotation of the flagella, resulting in a tumbling event (7, 15). Thus, high intracellular concentrations of phospho-CheY correlate with the tumbling motion of the cell. Smooth swimming is restored by dephosphorylation of CheY, which may be due to both the spontaneous hydrolysis of the phosphate group and the activity of the CheZ protein, which strongly accelerates the dephosphorylation of phospho-CheY (9, 11). It has also been established that phospho-CheY binds to the CheZ protein and that, following dephosphorylation, CheY is released from CheZ (4,18).CheZ is a ϳ24-kDa protein characterized in E. coli and S. typhimurium and does not seem to have significant homology with other bacterial proteins. Recently, a CheZ homolog has been described in Pseudomonas aeruginosa (13). It is able to complement an E. coli cheZ deletion strain. Proteins with analogous functions have been identified in other organisms. For example, Spo0E in Bacillus subtilis accelerates the dephosphorylation of the response regulator Spo0A (14); however, this phosphatase is not homologous to CheZ.The molecular mechanism through which CheZ...

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In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants

Sanna

1996

J Bacteriol

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“…Phosphorylated CheA transfers the phosphate group to the response regulator CheY (7,8), which in the phosphorylated form is able to interact with the flagellar switch and modify the sense of flagellar rotation from counterclockwise to clockwise, promoting tumble motion (9 -13). The CheZ protein accelerates the dephosphorylation of CheY, thus restoring smooth swimming (7,14,15).The mechanism of interaction between CheY and CheZ has not been elucidated, although some observations suggest an allosteric effect of CheZ on CheY instead of canonical phosphatase activity (16). We isolated a mutant CheY(N23D) that is resistant to the dephosphorylating activity of CheZ but is not impaired in phosphorylation or autodephosphorylation (16), which suggests that the mutant CheY may have a wild-type conformation but could be impaired in its ability to interact with CheZ.…”

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Isolation and in Vitro Characterization of CheZ Suppressors for the Escherichia coli Chemotactic Response Regulator Mutant CheYN23D

Sanna

1996

Journal of Biological Chemistry

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The phosphorylated form of the response regulator CheY promotes the tumble signal in Escherichia coli chemotaxis. Phospho-CheY is thought to interact with the switch at the base of the flagellar motor and cause reversal of flagellar rotation from counterclockwise to clockwise changing the swimming direction. Thus the level of phospho-CheY controls the direction of flagellar rotation. The decay of the tumble signal is caused by dephosphorylation of CheY. CheY has an intrinsic autophosphatase activity; however, this reaction is greatly accelerated by the presence of the CheZ protein.We have shown previously that mutations at residues Asn-23 and Lys-26 in CheY confer resistance to the dephosphorylation activity of CheZ (Sanna, M. G., Swanson, R. V., Bourret, R. B., and Simon, M. I. (1995) Mol. Microbiol. 15, 1069 -1079). Here we show that mutant CheY(N23D) is impaired in binding to CheZ, which provides a possible explanation for its resistance to the dephosphorylation activity of CheZ. Moreover, we isolated CheZ second-site suppressors of CheY(N23D), which restore both dephosphorylation and binding activity in a CheY(N23D) background. When the CheZ suppressor mutations are mapped, they are found in two clusters at the N and C termini of the CheZ protein which could define two regions of interaction with CheY. Furthermore, these regions may generate a surface in the folded three-dimensional structure of CheZ required for interaction with CheY.The ability of Escherichia coli to survive in different environments may be the result of its ability to swim toward favorable conditions using the rotation of 6 -8 flagella. This process is called bacterial chemotaxis and has been extensively studied in the past 30 years. When flagella rotate counterclockwise, they promote swimming in one direction for an extensive period of time (smooth swimming); reverse rotation (clockwise) causes tumbling of the cell, thus reorienting the swimming direction. The signal transduction pathway that regulates bacterial chemotaxis consists of the transmembrane receptor proteins Tar, Tsr, Trg, and Tap; the two-component system CheA/CheY; and the CheW and CheZ proteins (for reviews see Refs. 1-4). The external changes detected by the receptor are "communicated" to the histidine kinase CheA, which is able to modulate its autophosphorylation capacity (5, 6). Phosphorylated CheA transfers the phosphate group to the response regulator CheY (7,8), which in the phosphorylated form is able to interact with the flagellar switch and modify the sense of flagellar rotation from counterclockwise to clockwise, promoting tumble motion (9 -13). The CheZ protein accelerates the dephosphorylation of CheY, thus restoring smooth swimming (7,14,15).The mechanism of interaction between CheY and CheZ has not been elucidated, although some observations suggest an allosteric effect of CheZ on CheY instead of canonical phosphatase activity (16). We isolated a mutant CheY(N23D) that is resistant to the dephosphorylating activity of CheZ but is not impaired in phosphorylati...

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“…Mg 2ϩ is required in the phosphorylation and dephosphorylation reactions of wild-type CheY (5,21,33). However, Mg 2ϩ binding to CheY D13K is not detectable by either resonance shifts in 19 F NMR spectra of 4-fluorophenylalanine-labeled protein or by quenching of Trp 58 fluorescence (28,34).…”

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confidence: 99%

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

Meiying

Bourret

Volz

1997

Journal of Biological Chemistry

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An aspartate to lysine mutation at position 13 of the chemotaxis regulatory protein CheY causes a constitutive tumbly phenotype when expressed at high copy number in vivo even though the mutant protein is not phosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylation, so knowledge of its structure could explain the activation mechanism of CheY. The x-ray crystallographic structure of the CheY D13K mutant has been solved and refined at 2.3 Å resolution to an R-factor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg 2؉ -free structure, but there are localized changes within the active site. The side chain of lysine 13 blocks access to the active site, whereas its ⑀-amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspartate 57 is weakened, and the solvent structure is perturbed. Although the D13K mutant has the inactive conformation in the crystalline form, rearrangements in the active site appear to weaken the overall structure of that region, potentially creating a metastable state of the molecule. If a conformational change is required for signaling by CheY D13K, then it most likely proceeds dynamically, in solution.Cells continuously monitor various chemical and physical parameters in their surrounding environment and use this information to implement appropriate adaptive responses to changing conditions. This vital task is accomplished by a cascade of transient protein phosphorylation and dephosphorylation events arranged so that the flow of phosphoryl groups reflects environmental conditions. One ubiquitous class of signal transduction networks is the "two-component" regulatory systems that control diverse processes such as behavior, development, physiology, and virulence in bacteria, as well as adaptive processes in eukaryotes (for reviews, see Refs.

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Protein Phosphorylation and Bacterial Chemotaxis

Cited by 51 publications

References 0 publications

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants

Isolation and in Vitro Characterization of CheZ Suppressors for the Escherichia coli Chemotactic Response Regulator Mutant CheYN23D

Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis

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