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P Matsumura

Publications and source records attributed to P Matsumura.

At least 19 recordsLinked to original sources
PubMed2001

Crystallization and preliminary X-ray crystallographic analysis of FlhD/FlhC complex from Escherichia coli.

The heterotetrameric (C(2)D(2)) FlhD/FlhC complex was first discovered as a transcriptional activator of the flagellar genes in Escherichia coli. Recent studies now show that FlhD/FlhC also regulates several non-flagellar target genes in E. coli. The FlhD/FlhC complex also plays several important roles in other microorganisms. The molecular interactions between FlhD and FlhC, as well as the mechanisms by which the complex may vary its DNA-binding specificity, are not clear. Determination of the FlhD/FlhC crystal structure will provide insight into these protein-protein and protein-DNA interactions. The initial steps in this investigation are reported here: the overexpression, purification and crystallization of the FlhD/FlhC complex, the characterization of this crystal form and the recording and processing of an initial diffraction data set. The obtained crystal form of the FlhD/FlhC complex is hexagonal (space group P6(1), unit-cell parameters a = b = 150.5, c = 115.9 A). The crystal density is very low (V(M) = 5.5), with 81.7% of its volume occupied by solvent. A single C(2)D(2) tetramer is present in the crystallographic asymmetric unit. A complete native data set has been collected to 4.5 A resolution.

Crystallization↗
PubMed2001

FlhD/FlhC-regulated promoters analyzed by gene array and lacZ gene fusions.

The Escherichia coli transcriptional regulatory complex FlhD/FlhC, initially identified as a flagella-specific activator, is a global regulator involved in many cellular processes. Using gene arrays, lacZ gene fusions and enzyme assays, eight new targets of FlhD/FlhC were recognized. These are the transporter for galactose (MglBAC), the rod-shape determination proteins (MreBCD), malate dehydrogenase, and several enzymes involved in anaerobic respiration (glycerol 3-phosphate dehydrogenase, GlpABC; periplasmic nitrate reductase, NapFAGHBC; nitrite reductase, NrfABCDEFG; dimethyl sulfoxide reductase, DmsABC; and the modulator for hydrogenases, HydNHypF).

Bacterial Proteins↗
PubMed2001

Crystal structure of the global regulator FlhD from Escherichia coli at 1.8 A resolution.

FlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA-binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno-methionine for natural sulphur-methionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple-energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly alpha-helical. The C-termini of both FlhD monomers (residues 83-116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C-terminus structure in chain A (residues 83-98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA-binding, helix-turn-helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA-binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA-binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homodimers. Alanine substitution at Cys-65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.

Crystallization↗
PubMed2001

Extensive alanine scanning reveals protein-protein and protein-DNA interaction surfaces in the global regulator FlhD from Escherichia coli.

FlhD and FlhC are the transcriptional activators of the flagellar regulon. The heterotetrameric complex formed by these two proteins activates the transcription of the class II flagellar genes. The flagellar regulon consists not only of flagellar genes, but also of the chemotactic genes and some receptor proteins. Recently, a connection between the flagellar regulon and some virulence genes has been found in some species. Furthermore, FlhD, but not FlhC, regulates another non-flagellar target. As a first attempt to understand the mechanism of the flagellar transcriptional activation by FlhD and FlhC, the structure of FlhD has been solved. In order to understand the mechanism of the action of FlhD when it regulates the flagellar genes, we conducted site-directed mutagenesis based on its three-dimensional structure. Six interaction surfaces in the FlhD dimer were mapped by alanine scanning mutagenesis. Two of them are surface clusters formed by residues His-2, Asp-28, Arg-35, Phe-34 and Asn-61 located at each side of the dimer core. The other four are located in the flexible arms of the dimer. The residues Ser-82, Arg-83, Val-84, His-91, Thr-92, Ile-94 and Leu-96 are located at this region. All these residues are involved in the FlhD/FlhC interaction with the exception of Ser-82, Arg-83 and Val-84. These three residues affect the DNA-binding ability of the complex. The three-dimensional topology of FlhD and the site-directed mutagenesis results support the hypothesis of FlhC as an allosteric effector that activates FlhD for the recognition of the DNA.

Alanine↗
PubMed2000

The 1.9 A resolution crystal structure of phosphono-CheY, an analogue of the active form of the response regulator, CheY.

To structurally characterize the activated state of the transiently phosphorylated signal transduction protein CheY, we have constructed an alpha-thiophosphonate derivative of the CheY D57C point mutant and determined its three-dimensional structure at 1.85 A resolution. We have also characterized this analogue with high-resolution NMR and studied its binding to a peptide derived from FliM, CheY's target component of the flagellar motor. The chemically modified derivative, phosphono-CheY, exhibits many of the chemical properties of phosphorylated wild-type CheY, except that it is indefinitely stable. Electron density for the alpha-thiophosphonate substitution is clear and readily interpretable; omit refinement density at the phosphorus atom is greater than 10sigma. The molecule shows a number of localized conformational changes that are believed to constitute the postphosphorylation activation events. The most obvious of these changes include movement of the side chain of the active site base, Lys 109, and a predominately buried conformation of the side chain of Tyr 106. In addition, there are a number of more subtle changes more distant from the active site involving the alpha4 and alpha5 helices. These results are consistent with our previous structural interpretations of other CheY activation mutants, and with our earlier hypotheses concerning CheY activation through propagation of structural changes away from the active site.

Bacterial Proteins↗
PubMed1998

Synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis.

CheY is a signal transduction protein of the bacterial chemotaxis system that acts as a molecular switch to alter the swimming behavior of the bacterium. When CheY becomes phosphorylated at Asp57, CheY-Pi interacts with flagellar motor proteins, including FliM, to increase the likelihood that the flagellar motor will change its sense of rotation, increasing the frequency of tumbling. The structure of CheY in its dephosphorylated (inactive) state has been intensively investigated. The short lifetime ( approximately 20 s) of the aspartyl phosphate has precluded the complete structural determination of CheY-Pi. We have synthesized an analogue of CheY-Pi by alkylating an aspartate-to-cysteine mutant at position 57 of CheY to add a phosphonomethyl group at Cys57. This analogue, phosphono-CheY, is stable for months. Phosphono-CheY binds to two of the targets of CheY-Pi, FliM and CheZ, in a manner similar to that of CheY-Pi and much better than either unphosphorylated CheY or the unmodified form of D57C CheY. Phosphono-CheY also binds Mg(II) with a dissociation constant of approximately 6 mM at neutral pH and moderate salt level. These observations indicate that phosphono-CheY is a good biochemical analogue of CheY-Pi.

Alkylation↗
PubMed1998

Flagellar motor-switch binding face of CheY and the biochemical basis of suppression by CheY mutants that compensate for motor-switch defects in Escherichia coli.

CheY is a response regulator protein of Escherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis. The switch complex is composed of three proteins, FliG, FliM, and FliN. Recent biochemical data suggest a direct interaction of CheY with FliM. In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY with limited allele specificity. The protein products of suppressor cheY alleles were purified and assayed for FliM binding. Six out of nine CheY mutants were defective in FliM binding. Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY. To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding. Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants. This was not expected if CheY suppressors were compensatory conformational suppressors. Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains. However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic. Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called "pseudotaxis." Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment. The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY. Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes.

Bacterial Proteins↗
PubMed1998

Crystallization and preliminary X-ray analysis of FlhD from Escherichia coli.

The flhD gene from Escherichia coli codes for one of the transcriptional activators of flagellar genes and is a putative global regulator in the cell. FlhD together with FlhC forms a complex that positively regulates the expression of flagellar genes. However, FlhD is able to change its DNA binding specificity, probably by forming another complex with some unknown factor or by binding to other targets by itself. FlhD is a good model to study the DNA binding specificity by complex formation. The FlhD three-dimensional structure will certainly improve our understanding of this protein, which is the first case of combinatory specificity in prokaryotes. The flhD gene was over-expressed, and the resulting protein was purified to homogeneity. FlhD crystals were obtained from 15-25% PEG 5000, 0.05-0.1 M lithium sulfate, 0.1 M Tris, pH 8.5, or 20-30% PEG 5000, 0.05-0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5, as precipitant solutions, and belong to the space group I4 with unit cell parameters of a = b = 90 A, c = 42.6 A, and alpha = beta = gamma = 90 degrees and contain two molecules per asymmetric unit. A complete native data set has been collected to 2.3-A resolution.

Bacterial Proteins↗
PubMed1997

The CheZ-binding surface of CheY overlaps the CheA- and FliM-binding surfaces.

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 beta5-alpha5 loop, the alpha1-helix, and part of the alpha5-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 beta4-alpha4 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.

Bacterial Proteins↗
PubMed1997

Crystal structures of CheY mutants Y106W and T87I/Y106W. CheY activation correlates with movement of residue 106.

Position 106 in CheY is highly conserved as an aromatic residue in the response regulator superfamily. In the structure of the wild-type, apo-CheY, Tyr106 is a rotamer whose electron density is observed in both the inside and the outside positions. In the structure of the T87I mutant of CheY, the threonine to isoleucine change at position 87 causes the side chain of Tyr106 to be exclusively restricted to the outside position. In this report we demonstrate that the T87I mutation causes cells to be smooth swimming and non-chemotactic. We also show that another CheY mutant, Y106W, causes cells to be more tumbly than wild-type CheY, and impairs chemotaxis. In the structure of Y106W, the side chain of Trp106 stays exclusively in the inside position. Furthermore, a T87I/Y106W double mutant, which confers the same phenotype as T87I, restricts the side chain of Trp106 to the outside position. The results from these behavioral and structural studies indicate that the rotameric nature of the Tyr106 residue is involved in activation of the CheY molecule. Specifically, CheY's signaling ability correlates with the conformational heterogeneity of the Tyr106 side chain. Our data also suggest that these mutations affect the signal at an event subsequent to phosphorylation.

Bacterial Proteins↗
PubMed1997

The use of flash photolysis for a high-resolution temporal and spatial analysis of bacterial chemotactic behaviour: CheZ is not always necessary for chemotaxis.

The purpose of this work was to develop a high-resolution analysis of behaviour as an assay of the physiological consequences of mutations in the che genes and also to examine the role of CheZ in chemotaxis. Recent advances in flash photolysis have made it possible to expose cells to an unstable chemical gradient created by a square-wave increase in attractant concentration. The response of individual cells can be tracked in the order of milliseconds using real-time motion analysis. The tumble frequency of wild-type Escherichia coli exposed to photoreleased aspartate falls very quickly to smooth-swimming levels, and the swimming speed of these cells rises. As a consequence of these behavioural changes, there is an increase in the number of bacteria present in the centre of the flashed area, that is the bacteria's response to the transient gradient generated by flash photolysis was to swim into the centre of the flash area. This allowed the rapid quantitative measurement of chemotaxis. Deletion of various che genes resulted in predictable changes in chemotactic behaviour. cheZ null mutants are non-chemotactic when measured by classical techniques but demonstrate a definite chemotactic response to photoreleased attractant.

Bacterial Proteins↗
PubMed1997

Phosphorylating and dephosphorylating protein complexes in bacterial chemotaxis.

During optimal motility conditions, a 1:1 stoichiometry of CheA(L) (654 amino acids) to CheA(S) (557 amino acids) was determined. It was also found that CheZ binding to CheA(S) was inhibited by CheA(L)-CheA(S)-CheW interaction. This suggests that CheA(S) has different functions in the phosphorylating complex (CheA(L)-CheA(S)-CheW) and in the dephosphorylating complex (CheA(S)-CheZ).

Bacteria↗
PubMed1997

Cell cycle regulation of flagellar genes.

The expression of the flagellar master operon, flhDC, peaked in the middle of three consecutive cell cycles. The level of expression was lowest at the time of cell division. The expression of the second-level operon, flhB, peaked at cell division. The swimming speed of individual cells was also highest at the time of cell division.

Bacteria↗
PubMed1996

Characterization of the CheAS/CheZ complex: a specific interaction resulting in enhanced dephosphorylating activity on CheY-phosphate.

The cheA gene encodes two overlapping polypeptides with a common carboxyl terminus: CheAL and CheAS. CheAL plays a central role in the Escherichia coli chemotaxis signalling pathway by autophosphorylation and transferring the phosphate to both CheY and CheB. On the other hand, the physiological functions of CheAS remain unknown. We have observed that overproduction of CheAS in wild-type cells increased counterclockwise-biased flagellar rotation, and this effect is dependent on the presence of CheZ. CheZ specifically facilitates CheY-phosphate (CheY-P) dephosphorylation and generates a smooth swimming signal. A physical interaction was detected between CheZ and CheAS in wild-type cell lysates by immunoprecipitation. The CheAS/CheZ interaction does not require other chemotaxis components, as we could form the complex using purified CheAS and CheZ proteins. The ability of CheAS to bind to CheZ depends on its being in the reduced state. We found that under non-reducing conditions, CheAS appears to form intermolecular disulphide bonds and loses the ability to bind to CheZ. Finally, the CheAS/CheZ complex formed in vitro shows a greater dephosphorylating activity on CheY-P than does free CheZ.

Bacterial Proteins↗
PubMed1996

Differential regulation of multiple overlapping promoters in flagellar class II operons in Escherichia coli.

The Escherichia coli flagellar operons are divided into three categories: classes I, II and III. Expression of class II depends on expression of class I. One of the class II gene products, the FIIA protein, is an alternative sigma factor (sigma 28) required for transcription of the class III operons. In this study, we have characterized, in vitro, a role of sigma 28 in the regulation of the class II operons. Among the three class II operons examined, the fliA and fliL operons, but not the flhB operon, could be transcribed by both sigma 70 RNA polymerase holoenzyme with FihD/C (E sigma 70-FlhD/C) and sigma 28 RNA polymerase holoenzyme (E sigma 28). The flhB operon could only be transcribed by E sigma 70-FlhD/C under the conditions used. Both the fliA and fliL operons contained two overlapping promoters oriented in tandem. The transcription of fliA directed by E sigma 28 could outcompete that by E sigma 70-FlhD/C, indicating a positive autoregulation. However, E sigma 28 could not displace E sigma 70-FlhD/C bound to the fliL promoter. The sigma 28-mediated positive regulation of the class II operons involved a mechanism in which sigma 28 competed with sigma 70 for core RNA polymerase. In addition, recruitment of core RNA polymerase from the sigma 70 -10 site to the sigma 28 -10 was facilitated by formation of E sigma 70-FlhD/C pre-initiation complex. Taken together, the three class II promoters investigated are different in terms of their regulation by sigma 28. We propose that class II operons may be further divided into different subcategories.

Bacterial Proteins↗
PubMed1996

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies.

Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.

Bacterial Proteins↗
PubMed1996

Tyrosine 106 of CheY plays an important role in chemotaxis signal transduction in Escherichia coli.

CheY is the response regulator in the signal transduction pathway of bacterial chemotaxis. Position 106 of CheY is occupied by a conserved aromatic residue (tyrosine or phenylalanine) in the response regulator superfamily. A number of substitutions at position 106 have been made and characterized by both behavioral and biochemical studies. On the basis of the behavioral studies, the phenotypes of the mutants at position 106 can be divided into three categories: (i) hyperactivity, with a tyrosine-to-tryptophan mutation (Y106W) causing increased tumble signaling but impairing chemotaxis; (ii) low-level activity, with a tyrosine-to-phenylalanine change (Y106F) resulting in decreased tumble signaling and chemotaxis; and (iii) no activity, with substitutions such as Y106L, Y106I, Y106V, Y106G, and Y106C resulting in no chemotaxis and a smooth-swimming phenotype. All three types of mutants can be phosphorylated by CheA-phosphate in vitro to a level similar to that of wild-type CheY. Autodephosphorylation rates are similar for all categories of mutants. All mutant proteins displayed less than twofold increased rates compared with wild-type CheY. Binding of the mutant proteins to FliM was similar to that of the wild-type CheY in the CheY-FliM binding assays. The combined results from in vivo behavioral and in vitro biochemical studies suggest that the diverse phenotypes of the Y106 mutants are not due to a variation in phosphorylation or dephosphorylation ability nor in affinity for the switch. With reference to the structures of wild-type CheY and the T871 CheY mutant, our results suggest that rearrangements of the orientation of the tyrosine side chain at position 106 are involved in the signal transduction of CheY. These data also suggest that the binding of phosphoryl-CheY to the flagellar motor is a necessary, but not sufficient, event for signal transduction.

Bacterial Proteins↗