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Philip Matsumura - One of the best experts on this subject based on the ideXlab platform.
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The CheZ-binding Surface of CheY Overlaps the CheA- and FliM-binding Surfaces
The Journal of biological chemistry, 1997Co-Authors: Xiangyang Zhu, Karl Volz, Philip MatsumuraAbstract:Abstract CheY, the response regulator of bacterial chemotaxis, plays a pivotal role in signal transduction in bacterial chemotaxis and interacts with at least three Proteins: CheA, FliM, and CheZ. CheA receives signals from chemoreceptors and then transfers the signal to CheY by a phosphotransfer reaction. Phosphorylated CheY binds to FliM, one of the switch Proteins, resulting in a change in flagellar rotation from counterclockwise to clockwise. Phosphorylated CheY is dephosphorylated by its intrinsic autophosphatase activity and by CheZ. The CheA- and FliM-binding surfaces of CheY have been well studied, but characterization of the CheZ-binding surface of CheY is incomplete. We have analyzed the effect of CheZ on the dephosphorylation rates of 14 mutants of CheY. Nine mutant CheY Proteins showed more resistance to CheZ phosphatase activity than did wild-type CheY. These nine mutant CheY Proteins could be divided into two groups: one with altered CheZ binding and the other with normal CheZ binding. The mutations causing reduced CheZ binding altered residues on the same surface of CheY, a region consisting of the β5-α5 loop, the α1-helix, and part of the α5-helix. Mutations rendering CheY resistant to CheZ, isolated by Sanna et al. (Sanna, M. G., Swanson, R. V., Bourret, R. B., and Simon, M. I. (1995) Mol. Microbiol. 15, 1069–1079), were also found to affect this surface. The mutations in the CheY Protein that affect CheZ activity but not CheZ binding are located in the β4-α4 loop, which appears to be involved in the catalytic activity of CheZ. Finally, our results indicate that the CheY surfaces that bind CheA, FliM, and CheZ overlap, but are not completely identical.
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Signal transduction in chemotaxis. A propagating conformation change upon phosphorylation of CheY.
The Journal of biological chemistry, 1994Co-Authors: David F. Lowry, Amy F. Roth, P B Rupert, F W Dahlquist, F J Moy, P J Domaille, Philip MatsumuraAbstract:Abstract The CheY Protein from Escherichia coli and Salmonella typhimurium are among the best characterized Proteins of the receiver domain family of two component signal transduction systems in bacteria. Phosphorylation of CheY plays a central role in bacterial chemotaxis. However, it is not entirely clear how its state of phosphorylation contributes to its function. Genetic evidence suggests that CheY changes its conformation upon phosphorylation. We present evidence for this conformation change by comparing the NMR 15N-1H correlation spectra of CheY.Mg2+ complex and phospho-CheY in the presence of magnesium. Large changes in chemical shift are used as indicators of chemical changes and probable structural changes in the Protein backbone. Our observations suggest that significant structural changes occur in CheY upon phosphorylation and that these changes are distinct from the changes produced by magnesium ion binding. In addition to residues Asn-59 and Gly-65 that are immediately adjacent to the site of phosphorylation at Asp-57, a large number of other residues show significant chemical shift changes as a result of phosphorylation. These include Met-17, Val-21, Asn-23, Gly-39, Met-60, Met-63, Asp-64, Leu-66, Glu-67, Leu-68, Leu-69, Met-85, Val-86, Thr-87, Ala-88, Asn-94, Val-107, Lys-109, Thr-112, Ala-113, Ala-114, and Asn-121. These results appear inconsistent with the recent suggestion that phosphorylation produces the same structural changes as magnesium binding (Bellsolell, L., Prieto, J., Serrano, L., and Coll, M. (1994) J. Mol. Biol. 238, 489-495). We find that some regions change overlap with a genetically defined motor binding face. We therefore propose that the conformation switch modulates the interaction of CheY with its target, the flagellar motor. Other regions also change, possibly reflecting the many different functions of CheY homologues.
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A chemotactic signaling surface on CheY defined by suppressors of flagellar switch mutations.
Journal of bacteriology, 1992Co-Authors: S J Roman, Karl Volz, M Meyers, Philip MatsumuraAbstract:CheY is the response regulator Protein that interacts with the flagellar switch apparatus to modulate flagellar rotation during chemotactic signaling. CheY can be phosphorylated and dephosphorylated in vitro, and evidence indicates that CheY-P is the activated form that induces clockwise flagellar rotation, resulting in a tumble in the cell's swimming pattern. The flagellar switch apparatus is a complex macromolecular structure composed of at least three gene products, FliG, FliM, and FliN. Genetic analysis of Escherichia coli has identified fliG and fliM as genes in which mutations occur that allele specifically suppress CheY mutations, indicating interactions among these gene products. We have generated a class of CheY mutations selected for dominant suppression of fliG mutations. Interestingly, these CheY mutations dominantly suppressed both fliG and fliM mutations; this is consistent with the idea that the CheY Protein interacts with both switch gene products during signaling. Biochemical characterization of wild-type and suppressor CheY Proteins did not reveal altered phosphorylation properties or evidence for phosphorylation-dependent CheY multimerization. These data indicate that suppressor CheY Proteins are specifically altered in the ability to transduce chemotactic signals to the switch at some point subsequent to phosphorylation. Physical mapping of suppressor amino acid substitutions on the crystal structure of CheY revealed a high degree of spatial clustering, suggesting that this region of CheY is a signaling surface that transduces chemotactic signals to the switch.
Melvin I Simon - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of CheY from Thermotoga maritima do not support conventional explanations for the structural basis of enhanced thermostability.
Protein science : a publication of the Protein Society, 1998Co-Authors: K.c. Usher, Melvin I Simon, Frederick W. Dahlquist, A. F. A. De La Cruz, Ronald V. Swanson, S.j. RemingtonAbstract:The crystal structure of CheY Protein from Thermotoga maritima has been determined in four crystal forms with and without Mg++ bound, at up to 1.9 A resolution. Structural comparisons with CheY from Escherichia coli shows substantial similarity in their folds, with some concerted changes propagating away from the active site that suggest how phosphorylated CheY, a signal transduction Protein in bacterial chemotaxis, is recognized by its targets. A highly conserved segment of the Protein (the "y-turn loop," residues 55-61), previously suggested to be a rigid recognition determinant, is for the first time seen in two alternative conformations in the different crystal structures. Although CheY from Thermotoga has much higher thermal stability than its mesophilic counterparts, comparison of structural features previously proposed to enhance thermostability such as hydrogen bonds, ion pairs, compactness, and hydrophobic surface burial would not suggest it to be so.
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In vivo and in vitro characterization of Escherichia coli Protein CheZ gain- and loss-of-function mutants.
Journal of bacteriology, 1996Co-Authors: M G Sanna, Melvin I SimonAbstract: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.
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Activation of the phosphosignaling Protein CheY. II. Analysis of activated mutants by 19F NMR and Protein engineering.
The Journal of biological chemistry, 1993Co-Authors: Robert B. Bourret, Melvin I Simon, Steven K. Drake, Stephen A. Chervitz, Joseph J. FalkeAbstract:The Escherichia coli CheY Protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY Proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phosphorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational "switch" in the activation site, is required for activation of CheY by either phosphorylation or mutation. The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated Protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch.
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Conserved Aspartate Residues and Phosphorylation in Signal Transduction by the Chemotaxis Protein CheY
Proceedings of the National Academy of Sciences of the United States of America, 1990Co-Authors: Robert B. Bourret, J F Hess, Melvin I SimonAbstract:The CheY Protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY Proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY Proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY Protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY Protein is to stabilize a transient conformational change that can generate tumbling behavior.
Michael Eisenbach - One of the best experts on this subject based on the ideXlab platform.
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Effects of Phosphorylation, Mg2+, and Conformation of the Chemotaxis Protein CheY on Its Binding to the Flagellar Switch Protein FliM
Biochemistry, 1994Co-Authors: Martin Welch, Kenji Oosawa, Shin-ichi Aizawa, Michael EisenbachAbstract:CheY is the response regulator of bacterial chemotaxis. Previously, we showed that CheY binds to the flagellar switch Protein FliM and that this binding is increased upon phosphorylation of CheY [Welch, M., Oosawa, K., Aizawa, S.-I., & Eisenbach, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8787-8791]. Here, we demonstrate that it is the phosphorylated conformation of CheY, rather than the phosphate group itself, that is recognized and bound by FliM. We found that subsequent to the phosphorylation of CheY, Mg2+ was not required for the binding of CheY to FliM. However, phosphorylation of CheY did cause a change in the coordination properties of Mg2+ in the acid pocket of the Protein. This change in the coordination of Mg2+ required the presence of the absolutely conserved residue Lys109. When Lys109 was substituted by arginine, the resulting CheY Protein was unable to adopt an active conformation upon phosphorylation, and the Protein was not bound by FliM. Surprisingly, the CheY13DK mutant Protein, which is active in vivo but cannot be phosphorylated in vitro, exhibited only a low level of FliM binding activity, suggesting that its ability to cause clockwise rotation in the cell is not due to a constitutively high level of FliM binding. On the basis of these findings, we propose a mechanism for CheY activation by phosphorylation.
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Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Martin Welch, Kenji Oosawa, Shin-ichi Aizawa, Michael EisenbachAbstract:Abstract Regulation of the direction of flagellar rotation is central to the mechanism of bacterial chemotaxis. The transitions between counterclockwise and clockwise rotation are controlled by a "switch complex" composed of three Proteins (FliG, FliM, and FliN) and located at the base of the flagellar motor. The mechanism of function of the switch is unknown. Here we demonstrate that the diffusible clockwise-signal molecule, the CheY Protein, binds to the switch, that the primary docking site is FliM, that the extent of CheY binding to FliM is dependent upon the phosphorylation level of CheY, and that it is unaffected by the other two switch Proteins. This study provides a biochemical demonstration of binding of a signal molecule to the bacterial switch and demonstrates directly that phosphorylation regulates the activity of this molecule.
Robert B. Bourret - One of the best experts on this subject based on the ideXlab platform.
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Throwing the switch in bacterial chemotaxis.
Trends in microbiology, 1999Co-Authors: Ruth E. Silversmith, Robert B. BourretAbstract:In Escherichia coli chemotaxis, the switch from counterclockwise to clockwise rotation of the flagella occurs as a result of binding of the phosphorylated CheY Protein to the base of the flagellum. Analysis of CheY variants has provided a picture of the surface of CheY that undergoes conformational shifts, as a result of phosphorylation, to interact directly with the flagellum. Whether phospho-CheY binding and flagellar switching are sequential steps or can occur in a concerted fashion has yet to be determined.
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Purification and Characterization of Bacillus subtilis CheY
Biochemistry, 1993Co-Authors: David S. Bischoff, Robert B. Bourret, M. L. Kirsch, George W. OrdalAbstract:Amino acid sequence comparison suggests that numerous Proteins are common to the signal transduction pathways controlling chemotaxis in Bacillus subtilis and Escherichia coli. However, previous work has indicated several differences between the two systems. We have undertaken a comparative study of the roles of the CheY Protein in chemotaxis by B. subtilis and E. coli. Although CheY from the two species share only 36% amino acid sequence identity, purified B. subtilis CheY was phosphorylated in vitro by E. coli CheA, and dephosphorylation of CheY-P was enhanced by E. coli CheZ. Alteration of the putative site of phosphorylation in B. subtilis CheY, Asp54, eliminated chemotaxis in vivo, further confirming that phosphorylation is important for B. subtilis chemotaxis. Loss of CheY function resulted in tumbling behavior in B. subtilis. Introduction of positively charged residues in place of Asp10 of B. subtilis CheY abolished function, whereas the corresponding changes in E. coli CheY apparently result in constitutive activation. The B. subtilis CheY Asp10 mutant Proteins also failed to cause tumbling in E. coli, consistent with a different interaction between CheY and the flagellar switch in the two species. Finally, B. subtilis adapted more rapidly to positive stimuli than negative stimuli, whereas the opposite is true of E. coli. We conclude that B. subtilis regulates its response to positive chemotactic stimuli by enhancing phosphorylation of chemotaxis Proteins, whereas E. coli reduces phosphorylation in the same circumstance.
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Activation of the phosphosignaling Protein CheY. II. Analysis of activated mutants by 19F NMR and Protein engineering.
The Journal of biological chemistry, 1993Co-Authors: Robert B. Bourret, Melvin I Simon, Steven K. Drake, Stephen A. Chervitz, Joseph J. FalkeAbstract:The Escherichia coli CheY Protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY Proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phosphorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational "switch" in the activation site, is required for activation of CheY by either phosphorylation or mutation. The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081-13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation. (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated Protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch.
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Conserved Aspartate Residues and Phosphorylation in Signal Transduction by the Chemotaxis Protein CheY
Proceedings of the National Academy of Sciences of the United States of America, 1990Co-Authors: Robert B. Bourret, J F Hess, Melvin I SimonAbstract:The CheY Protein is phosphorylated by CheA and dephosphorylated by CheZ as part of the chemotactic signal transduction pathway in Escherichia coli. Phosphorylation of CheY has been proposed to occur on an aspartate residue. Each of the eight aspartate residues of CheY was replaced by using site-directed mutagenesis. Substitutions at Asp-12, Asp-13, or Asp-57 resulted in loss of chemotaxis. Most of the mutant CheY Proteins were still phosphorylated by CheA but exhibited modified biochemical properties, including reduced ability to accept phosphate from CheA, altered phosphate group stability, and/or resistance to CheZ-mediated dephosphorylation. The properties of CheY Proteins bearing a substitution at position 57 were most aberrant, consistent with the hypothesis that Asp-57 is the normal site of acyl phosphate formation. Evidence for an alternate site of phosphorylation in the Asp-57 mutants is presented. Phosphorylated CheY is believed to cause tumbling behavior. However, a dominant mutant CheY Protein that was not phosphorylated in vitro caused tumbling in vivo in the absence of CheA. This phenotype suggests that the role of phosphorylation in the wild-type CheY Protein is to stabilize a transient conformational change that can generate tumbling behavior.
Prasad Dhurjati - One of the best experts on this subject based on the ideXlab platform.
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Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.
Applied and environmental microbiology, 1995Co-Authors: Jim Klein, Prasad DhurjatiAbstract:The tendency of recombinant Protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between Protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of Protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant Protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY Protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of Protein aggregation in vivo, i.e., inclusion body formation, are discussed.