Aspartate Chemoreceptor Research Articles

Conformational suppression of inter-receptor signaling defects

Motile bacteria follow gradients of attractant and repellent chemicals with high sensitivity. Their chemoreceptors are physically clustered, which may enable them to function as a cooperative array. Although native chemoreceptor molecules are typically transmembrane homodimers, they appear to associate through their cytoplasmic tips to form trimers of dimers, which may be an important architectural element in the assembly and operation of receptor clusters. The five receptors of Escherichia coli that mediate most of its chemotactic and aerotactic behaviors have identical trimer contact residues and have been shown by in vivo crosslinking methods to form mixed trimers of dimers. Mutations at the trimer contact sites of Tsr, the serine chemoreceptor, invariably abrogate Tsr function, but some of those lesions (designated Tsr*) are epistatic and block the function of heterologous chemoreceptors. We isolated and characterized mutations (designated Tar()) in the aspartate chemoreceptor that restored function to Tsr* receptors. The suppressors arose at or near the Tar trimer contact sites and acted in an allele-specific fashion on Tsr* partners. Alone, many Tar() receptors were unable to mediate chemotactic responses to aspartate, but all formed clusters with varying efficiencies. Most of those Tar() receptors were epistatic to WT Tsr, but some regained Tar function in combination with a suppressible Tsr* partner. Tar()-Tsr* suppression most likely occurs through compensatory changes in the conformation or dynamics of a mixed receptor signaling complex, presumably based on trimer-of-dimer interactions. These collaborative teams may be responsible for the high-gain signaling properties of bacterial chemoreceptors.

Differential Recognition of Citrate and a Metal-Citrate Complex by the Bacterial Chemoreceptor Tcp

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metal-citrate sensing, and the mutated residues are located in or near the alpha1-alpha2 and alpha3-alpha4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn(67), which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn(67) and allosteric switching.

Helical distribution of the bacterial chemoreceptor via colocalization with the Sec protein translocation machinery

In Escherichia coli, chemoreceptor clustering at a cell pole seems critical for signal amplification and adaptation. However, little is known about the mechanism of localization itself. Here we examined whether the aspartate chemoreceptor (Tar) is inserted directly into the polar membrane by using its fusion to green fluorescent protein (GFP). After induction of Tar–GFP, fluorescent spots first appeared in lateral membrane regions, and later cell poles became predominantly fluorescent. Unexpectedly, Tar–GFP showed a helical arrangement in lateral regions, which was more apparent when a Tar–GFP derivative with two cysteine residues in the periplasmic domain was cross-linked to form higher oligomers. Moreover, similar distribution was observed even when the cytoplasmic domain of the double cysteine Tar–GFP mutant was replaced by that of the kinase EnvZ, which does not localize to a pole. Observation of GFP–SecE and a translocation-defective MalE–GFP mutant, as well as indirect immunofluorescence microscopy on SecG, suggested that the general protein translocation machinery (Sec) itself is arranged into a helical array, with which Tar is transiently associated. The Sec coil appeared distinct from the MreB coil, an actin-like cytoskeleton. These findings will shed new light on the mechanisms underlying spatial organization of membrane proteins in E. coli.

Osmotic stress mechanically perturbs chemoreceptors in Escherichia coli

Two-component signaling systems play a major role in the long-term adaptation of microorganisms to changes in osmolarity, but how osmoreceptors work is not well understood. Temporal changes in solute concentration are sensed by the chemotaxis system in Escherichia coli, enabling these bacteria to avoid regions of high osmolarity. To study how osmolarity is detected in this system, we fused yellow fluorescent protein (YFP) to the C terminus of the serine or aspartate chemoreceptor, monitored the steady-state fluorescence polarization of YFP, and found that the polarization decreased substantially upon addition of osmotic agents. This decrease was due to an increase in fluorescence resonance energy transfer between YFP fluorophores in adjacent homodimers within trimers of dimers. Thus, changes in homodimer spacing and/or orientation appear to initiate osmotactic signaling.

Stabilization of Polar Localization of a Chemoreceptor via Its Covalent Modifications and Its Communication with a Different Chemoreceptor

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.

Four-helix bundle: a ubiquitous sensory module in prokaryotic signal transduction.

Transmembrane chemoreceptors in Escherichia coli utilize ligand-binding domains for detecting various external signals. The structure of this domain in the E.coli aspartate receptor, Tar, is known and its signal transduction mechanism is under investigation. Current domain models for this important sensory module are inaccurate and, therefore, cannot reveal the distribution of this domain within the current genomic landscape. We carried out sensitive and exhaustive PSI-BLAST searches initiated with the sequence corresponding to a known structure of the four-helix, ligand-binding domain of the aspartate chemoreceptor. From the resulting sequences, we built a multiple sequence alignment for this domain family, which confirmed that the current TarH model is erroneous and fails to detect most of the domain homologs. In the process, we developed a technique that visualizes the secondary structure prediction of each protein sequence in order to improve the multiple sequence alignment. We found that the four-helix up-and-down bundle represents a large domain family and includes representatives of all major classes of prokaryotic signal transduction, namely histidine kinases, di-guanylate cyclases and chemotaxis receptors.

The MreB and Min cytoskeletal‐like systems play independent roles in prokaryotic polar differentiation

Establishment of an axis of cell polarity and differentiation of the cell poles are fundamental aspects of cellular development in many organisms. We compared the effects of two bacterial cytoskeletal-like systems, the MreB and MinCDE systems, on these processes in Escherichia coli. We report that the Min proteins are capable of establishing an axis of oscillation that is the initial step in establishment of polarity in spherical cells, in a process that is independent of the MreB cytoskeleton. In contrast, the MreB system is required for establishment of the rod shape of the cell and for polar targeting of other polar constituents, such as the Shigella virulence factor IcsA and the aspartate chemoreceptor Tar, in a process that is independent of the Min system. Thus, the two bacterial cytoskeletal-like systems act independently on different aspects of cell polarization.

Attractant binding alters arrangement of chemoreceptor dimers within its cluster at a cell pole.

Many sensory systems involve multiple steps of signal amplification to produce a significant response. One such mechanism may be the clustering of transmembrane receptors. In bacterial chemotaxis, where a stoichiometric His-Asp phosphorelay from the kinase CheA to the response regulator CheY plays a central role, the chemoreceptors (methyl-accepting chemotaxis proteins) cluster together with CheA and the adaptor CheW, at a pole of a rod-shaped cell. This clustering led to a proposal that signal amplification occurs through an interaction between chemoreceptor homodimers. Here, by using in vivo disulfide crosslinking assays, we examined an interdimer interaction of the aspartate chemoreceptor (Tar). Two cysteine residues were introduced into Tar: one at the subunit interface and the other at the external surface of the dimer. Crosslinked dimers and higher oligomers (especially a deduced hexamer) were detected and their abundance depended on CheA and CheW. The ligand aspartate significantly reduced the amounts of higher oligomers but did not affect the polar localization of Tar-GFP. Thus, the binding of aspartate alters the rate of collisions between Tar dimers in assembled signaling complexes, most likely due to a change in the relative positions or trajectories of the dimers. These collisions could occur within a trimer-ofdimers predicted by crystallography, or between such trimers. These results are consistent with the proposal that the interaction of chemoreceptor dimers is involved in signal transduction.

大腸菌アスパラギン酸レセプターTarの極クラスターにおけるダイマー間相互作用とシグナル増幅

大腸菌アスパラギン酸レセプターTarの極クラスターにおけるダイマー間相互作用とシグナル増幅

Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase.

In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation. The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors. The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability. In this study, six independent intragenic suppressors of the mutation were isolated. They were divided into two classes. Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA. These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate. Some other CheA-activating mutations were also found to serve as class I suppressors. These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor. The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif. This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar. This effect was reversed by introducing a mutation in the xWxxF motif. These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

Attractant Regulation of the Aspartate Receptor−Kinase Complex: Limited Cooperative Interactions between Receptors and Effects of the Receptor Modification State

The manner by which the bacterial chemotaxis system responds to a wide range of attractant concentrations remains incompletely understood. In principle, positive cooperativity between chemotaxis receptors could explain the ability of bacteria to respond to extremely low attractant concentrations. By utilizing an in vitro receptor-coupled kinase assay, the attractant-dependent response curve has been measured for the Salmonella typhimurium aspartate chemoreceptor. The attractant chosen, alpha-methyl aspartate, was originally used to quantitate high receptor sensitivity at low attractant concentrations by Segall, Block, and Berg [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8987-8991]. The attractant response curve exhibits limited positive cooperativity, yielding a Hill coefficient of 1.7-2.4, and this Hill coefficient is relatively independent of both the receptor modification state and the mole ratio of CheA to receptor. These results disfavor models in which there are strong cooperative interactions between large numbers of receptor dimers in an extensive receptor array. Instead, the results are consistent with cooperative interactions between a small number of coupled receptor dimers. Because the in vitro receptor-coupled kinase assay utilizes higher than native receptor densities arising from overexpression, the observed positive cooperativity may overestimate that present in native receptor populations. Such positive cooperativity between dimers is fully compatible with the negative cooperativity previously observed between the two symmetric ligand binding sites within a single dimer. The attractant affinity of the aspartate receptor is found to depend on the modification state of its covalent adaptation sites. Increasing the the level of modification decreases the apparent attractant affinity at least 10-fold in the in vitro receptor-coupled kinase assay. This observation helps explain the ability of the chemotaxis pathway to respond to a broad range of attractant concentrations in vivo.

The aspartate chemoreceptor Tar is effectively methylated by binding to the methyltransferase mainly through hydrophobic interaction.

In the chemotaxis of Escherichia coli, adaptation requires the methylation and demethylation of transmembrane receptors, which are catalysed by the methyltransferase CheR and the methylesterase CheB respectively. CheR binds to major chemoreceptors through their C-terminal motif NWETF, which is distinct from the methylation sites. In this study, we carried out a systematic mutagenesis of the pentapeptide sequence of Tar. Receptor methylation and adaptation were severely impaired by the alanine substitution of residue W550 and, to a lesser extent, by that of F553. Substitution of residues N549, E551 and T552 had only a slight or little effect. The defects of the W550A and F553A mutations were suppressed by high- and low-level overproduction of CheR respectively. Expression of a fusion protein containing the NWETF sequence, but not its W550A and F553A versions, inhibited chemotaxis of the Che+ strain. In an in vitro assay, CheR bound to the wild-type version but not to the mutant versions. These results and further mutagenesis suggest that the hydrophobicity and the size of residues W550 and F553 are critical in the interaction with CheR, a conclusion that is consistent with the crystal structure of a CheR-NWETF complex. On the other hand, the negatively charged side chain of E551 and the polar side chains of N549 and T552 may not be strictly required, although the presence of a salt bridge and hydrogen bonds between these residues and residues from CheR has been noted in the co-crystal.

Conversion of a bacterial warm sensor to a cold sensor by methylation of a single residue in the presence of an attractant.

The aspartate chemoreceptor (Tar) of Escherichia coli also serves as a thermosensor, and it is very amenable to genetic and biochemical analysis of the thermosensing mechanism. Its thermosensing properties are controlled by reversible methylation of the cytoplasmic signalling/adaptation domain of the protein. The unmethylated and the fully methylated (aspartate-bound) receptors sense, as attractant stimuli, increases (warm sensor) and decreases (cold sensor) in temperature respectively. To learn more about the mechanism of thermosensing, we replaced the four methyl-accepting glutamyl residues with non-methylatable aspartyl residues in all possible combinations. In a strain defective in both methyltransferase (CheR) and methylesterase (CheB) activities, all of the mutant Tar proteins functioned as warm sensors. To create a situation in which all of the remaining glutamyl residues were methylated, we expressed the mutant proteins in a CheB-defective, CheR-overproducing strain. The fully glutamyl-methylated proteins were designed to mimic the full range of methylation states possible for wild-type Tar. Almost all of the methylated mutant receptors, including those with single glutamyl residues, were cold sensors in the presence of aspartate. Thus, binding of aspartate to Tar and methylation of its single glutamyl residue can invert its temperature-dependent signalling properties.

Inversion of thermosensing property of the bacterial receptor tar by mutations in the second transmembrane region

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate α-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190–210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar.

1PA081 キメラレセプターを用いたサルモネラ菌化学感覚レセプターTcpのリガンド認識に関与する領域の解析

1PA081 キメラレセプターを用いたサルモネラ菌化学感覚レセプターTcpのリガンド認識に関与する領域の解析

Intersubunit Interaction between Transmembrane Helices of the Bacterial Aspartate Chemoreceptor Homodimer

The transmembrane domain that connects the extracellular and intracellular domains of cell-surface receptors must play a critical role in signal transduction. Here, we report studies of the interaction between the transmembrane helices (TM1 and TM2) of the Escherichia coli aspartate chemoreceptor (Tar). Tar exists as a homodimer regardless of its state of ligand occupancy. A particular residue substitution in TM1 (A19K) abolishes the signaling ability of Tar. This signaling defect can be suppressed by single residue substitutions in TM2 (W192R, A198E, V201E, and V202L). We have found that these suppressors can be divided into two groups. A198E and V201E (class 1) almost completely suppress the defects caused by A19K, and this suppression occurs between two subunits of the Tar dimer. In contrast, W192R and V202L (class 2) fail to suppress some signaling defects, and their suppression does not occur between subunits. Because disulfide-crosslinking studies predict that residues 198 and 201 point toward residue 19 of the partner subunit, we propose that the class 1 suppressors form an intersubunit salt bridge with Lys-19. Indeed, A19K was suppressed by several additional aspartate or glutamate substitutions on the same face of TM2 occupied by residues 198 and 201. None of these intersubunit salt bridges perturb signaling function, suggesting that the mechanism of transmembrane signal propagation does not involve large displacements (such as extensive rotation) of the TM1 and TM2 helices relative to each other.

Cysteine and disulfide scanning reveals two amphiphilic helices in the linker region of the aspartate chemoreceptor.

The transmembrane aspartate receptor of E. coli and S. typhimurium mediates cellular chemotaxis toward aspartate by regulating the activity of the cytoplasmic histidine kinase, CheA. Ligand binding results in transduction of a conformational signal through the membrane to the cytoplasmic domain where both kinase regulation and adaptation occur. Of particular interest is the linker region, E213 to Q258, which connects and transduces the conformational signal between the cytoplasmic end of the transmembrane signaling helix (alpha 4/TM2) and the major methylation helix of the cytoplasmic domain (alpha 6). This linker is crucial for stable folding and function of the homodimeric receptor. The present study uses cysteine and disulfide scanning mutagenesis to investigate the secondary structure and packing surfaces within the linker region. Chemical reactivity assays reveal that the linker consists of three distinct subdomains: two alpha-helices termed alpha 4 and alpha 5 and, between them, an ordered region of undetermined secondary structure. When cysteine is scanned through the helices, characteristic repeating patterns of solvent exposure and burial are observed. Activity assays, both in vivo and in vitro, indicate that each helix possesses a buried packing face that is crucial for proper receptor function. The interhelical subdomain is at least partially buried and is also crucial for proper receptor function. Disulfide scanning places helix alpha 4 distal to the central axis of the homodimer, while helix alpha 5 is found to lie at the subunit interface. Finally, sequence alignments suggest that all three linker subdomains are highly conserved among the large subfamily of histidine kinase-coupled sensory receptors that possess methylation sites for use in covalent adaptation.

Direct measurement of small ligand-induced conformational changes in the aspartate chemoreceptor using EPR.

Ligand-binding-induced conformational changes in the Salmonella typhimurium aspartate receptor were studied using spin-labeling electron paramagnetic resonance. Cysteine residues, introduced by site-directed mutagenesis at several positions in the aspartate receptor periplasmic domain, were used to attach covalently a thiol-specific spin label. The electron paramagnetic resonance spectra of these labeled proteins were obtained in the presence and absence of the ligand aspartate, and used to calculate the distance change between spin labels. The results support a model in which transmembrane signaling is executed by a combined movement of alpha helix 4 (which leads into transmembrane domain 2) relative to alpha helix 1 (connected to transmembrane domain 1), as well as a coming together of the two subunits. Ligand binding causes spin labels at position 39 and 179 (within one subunit) to move further from each other and spin labels at position 39 and 39' (between two subunits) to move closer to each other. Both of these changes are very small-less than 2.5 A. No similar changes were detected in any aspartate receptor samples solubilized in detergent, suggesting that the membrane is required for these conformational changes. This is the first case of physically measured ligand-induced changes in a full-length 1-2 transmembrane domain receptor, and the results suggest that very small ligand-induced movements can result in large effects on the activity of downstream proteins.

Thermosensing properties of mutant aspartate chemoreceptors with methyl-accepting sites replaced singly or multiply by alanine.

The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modification of its four methylation sites (Gln295, Glu302, Gln309, and Glu491). Without posttranslational deamidation, Tar has no thermosensing ability. When Gln295 and Gln309 are deamidated to Glu, the unmethylated and heavily methylated forms function as warm and cold sensors, respectively. In this study, we carried out alanine-scanning mutagenesis of the methylation sites. Although alanine substitutions influenced the signaling bias and the methylation level, all of the mutants retained aspartate-sensing function. Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function. In the posttranslational modification-defective background, some of the alanine substitutions restored thermosensing ability. Warm sensors were found among mutants retaining two glutamate residues, and cold sensors were found among those with one or no glutamate residue. This result suggests that the negative charge at the methylation sites is one factor that determines thermosensor phenotypes, although the size and shape of the side chain may also be important. The warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes were found. Thus, the different thermosensing phenotypes that result from covalent modification of the methylation sites may reflect distinct structural states. Broader implications for the thermosensing mechanism are also discussed.

Signaling by the Escherichia coli aspartate chemoreceptor Tar with a single cytoplasmic domain per dimer.

Many transmembrane receptors are oligomeric proteins. Binding of a ligand may alter the oligomeric state of the receptor, induce structural changes within the oligomer, or both. The bacterial aspartate chemoreceptor Tar forms a homodimer in the presence or absence of ligands. Tar mediates attractant and repellent responses by modulating the activity of the cytoplasmic kinase CheA. In vivo intersubunit suppression was used to show that certain combinations of full-length and truncated mutant Tar proteins complemented each other to restore attractant responses to aspartate. These results suggest that heterodimers with only one intact cytoplasmic domain are functional. The signaling mechanism may require interactions between dimers or conformational changes within a single cytoplasmic domain.