Chemotaxis in Rhodobacter sphaeroides requires an atypical histidine protein kinase

Rhodobacter sphaeroides has a complex chemosensory system comprising two classic CheAs, two atypical CheAs, and eight response regulators (six CheYs and two CheBs). The classic CheAs, CheA(1) and CheA(2), have similar domain structures to Escherichia coli CheA, whereas the atypical CheAs, CheA(3) and CheA(4), lack some of the domains found in E. coli CheA. CheA(2), CheA(3), and CheA(4) are all essential for chemotaxis. Here we demonstrate that CheA(3) and CheA(4) are both unable to undergo ATP-dependent autophosphorylation, however, CheA(4) is able to phosphorylate CheA(3). The in vitro kinetics of this phosphorylation reaction were consistent with a reaction mechanism in which CheA(3) associates with a CheA(4) dimer forming a complex, CheA(3)A(4). To the best of our knowledge, CheA(3)A(4) is the first characterized histidine protein kinase where the subunits are encoded by distinct genes. Selective phosphotransfer was observed from CheA(3)-P to the response regulators CheY(1), CheY(6), and CheB(2). Using phosphorylation site and kinase domain mutants of CheA we show that phosphosignaling involving CheA(2), CheA(3), and CheA(4) is essential for chemotaxis in R. sphaeroides. Interestingly, CheA(3) was not phosphorylated in vitro by CheA(1) or CheA(2), although CheA(1) and CheA(2) mutants with defective kinase domains were phosphorylated by CheA(4). Because in vivo CheA(3) and CheA(4) localize to the cytoplasmic chemotaxis cluster, while CheA(2) localizes to the polar chemotaxis cluster, it is likely that the physical separation of CheA(2) and CheA(4) prevents unwanted cross-talk between these CheAs.     

Mutations that affect control of the methylesterase activity of CheB, a component of the chemotaxis adaptation system in Escherichia coli.

Sensory adaptation by the chemotaxis system of Escherichia coli requires adjustments of the extent of methyl esterification of the chemotaxis receptor proteins. One mechanism utilized by E. coli to make such adjustments is to control the activity of CheB, the enzyme responsible for removing receptor methyl ester groups. Previous work has established the existence of a multicomponent signal transduction pathway that enables the chemotaxis receptor proteins to control the methylesterase activity in response to chemotactic stimuli. We isolated and characterized CheB mutants that do not respond normally to this control mechanism. In intact cells these CheB variants could not be activated in response to negative chemotaxis stimuli. Further characterization indicated that these CheB variants could not be phosphorylated by the chemotaxis protein kinase CheA. Disruption of the mechanism responsible for regulating methylesterase activity was also observed in cells carrying chromosomal deletions of either cheA or cheW as well as in cells expressing mutant versions of CheA that lacked kinase activity. These results provide further support for recent proposals that activation of the methylesterase activity of CheB involves phosphorylation of CheB by CheA. Furthermore, our findings suggest that CheW plays an essential role in enabling the chemotaxis receptor proteins to control the methylesterase activity, possibly by controlling the CheA-CheB phosphotransfer reaction.     

Cellular stoichiometry of the chemotaxis proteins in Bacillus subtilis

The chemoreceptor-CheA kinase-CheW coupling protein complex, with ancillary associated proteins, is at the heart of chemotactic signal transduction in bacteria. The goal of this work was to determine the cellular stoichiometry of the chemotaxis signaling proteins in Bacillus subtilis. Quantitative immunoblotting was used to determine the total number of chemotaxis proteins in a single cell of B. subtilis. Significantly higher levels of chemoreceptors and much lower levels of CheA kinase were measured in B. subtilis than in Escherichia coli. The resulting cellular ratio of chemoreceptor dimers per CheA dimer in B. subtilis is roughly 23.0 ± 4.5 compared to 3.4 ± 0.8 receptor dimers per CheA dimer observed in E. coli, but the ratios of the coupling protein CheW to the CheA dimer are nearly identical in the two organisms. The ratios of CheB to CheR in B. subtilis are also very similar, although the overall levels of modification enzymes are higher. When the potential binding partners of CheD are deleted, the levels of CheD drop significantly. This finding suggests that B. subtilis selectively degrades excess chemotaxis proteins to maintain optimum ratios. Finally, the two cytoplasmic receptors were observed to localize among the other receptors at the cell poles and appear to participate in the chemoreceptor complex. These results suggest that there are many novel features of B. subtilis chemotaxis compared with the mechanism in E. coli, but they are built on a common core.     

Phosphotransfer in Rhodobacter sphaeroides chemotaxis

The two-component sensing system controlling bacterial chemotaxis is one of the best studied in biology. Rhodobacter sphaeroides has a complex chemosensory pathway comprising two histidine protein kinases (CheAs) and eight downstream response regulators (six CheYs and two CheBs) rather than the single copies of each as in Escherichia coli. We used in vitro analysis of phosphotransfer to start to determine why R.sphaeroides has these multiple homologues. CheA(1) and CheA(2) contain all the key motifs identified in the histidine protein kinase family, except for conservative substitutions (F-L and F-I) within the F box of CheA(2), and both are capable of ATP-dependent autophosphorylation. While the K(m) values for ATP of CheA(1) and CheA(2) were similar to that of E.coli, the k(cat) value was three times lower, but similar to that measured for the related Sinorhizobium meliloti CheA. However, the two CheAs differed both in their ability to phosphorylate the various response regulators and the rates of phosphotransfer. CheA(2) phosphorylated all of the CheYs and both CheBs, whilst CheA(1) did not phosphorylate either CheB and phosphorylated only the response regulators encoded within its own genetic locus (CheY(1), CheY(2), and CheY(5)) and CheY(3). The dephosphorylation rates of the R.sphaeroides CheBs were much slower than the E.coli CheB. The dephosphorylation rate of CheY(6), encoded by the third chemosensory locus, was ten times faster than that of the E.coli CheY. However, the dephosphorylation rates of the remaining R.sphaeroides CheYs were comparable to that of E.coli CheY.     

Rapid phosphotransfer to CheY from a CheA protein lacking the CheY-binding domain

The histidine protein kinase CheA plays a central role in the bacterial chemotaxis signal transduction pathway. Autophosphorylated CheA passes its phosphoryl group to CheY very rapidly (k(cat) approximately 750 s(-)(1)). Phospho-CheY in turn influences the direction of flagellar rotation. The autophosphorylation site of CheA (His(48)) resides in its N-terminal P1 domain. The adjacent P2 domain provides a high-affinity binding site for CheY, which might facilitate the phosphotransfer reaction by tethering CheY in close proximity to the phosphodonor located in P1. To explore the contribution of P2 to the CheA --> CheY phosphotransfer reaction in the Escherichia coli chemotaxis system, we examined the transfer kinetics of a mutant CheA protein (CheADeltaP2) in which the 98 amino acid P2 domain had been replaced with an 11 amino acid linker. We used rapid-quench and stopped-flow fluorescence experiments to monitor phosphotransfer to CheY from phosphorylated wild-type CheA and from phosphorylated CheADeltaP2. The CheADeltaP2 reaction rates were significantly slower and the K(m) value was markedly higher than the corresponding values for wild-type CheA. These results indicate that binding of CheY to the P2 domain of CheA indeed contributes to the rapid kinetics of phosphotransfer. Although phosphotransfer was slower with CheADeltaP2 (k(cat)/K(m) approximately 1.5 x 10(6) M(-)(1) s(-)(1)) than with wild-type CheA (k(cat)/K(m) approximately 10(8) M(-)(1) s(-)(1)), it was still orders of magnitude faster than the kinetics of CheY phosphorylation by phosphoimidazole and other small molecule phosphodonors (k(cat)/K(m) approximately 5-50 M(-)(1) s(-)(1)). We conclude that the P1 domain of CheA also makes significant contributions to phosphotransfer rates in chemotactic signaling.     

Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli.

CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli. CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation. Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells. To find out why, we examined the effects of a purified P1 fragment, CheA[1-149], on CheA-related signaling activities in vitro and devised in vivo assays for those same activities. Although readily phosphorylated by CheA[260-537], the CheA catalytic domain, CheA[1-149], was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center. CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA[1-149], suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect. In agreement with these findings, a 40-fold excess of CheA[1-149] fragments did not impair the CheA autophosphorylation reaction. CheA[1-149] did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules. An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site. The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells. However, an excess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragments have both transphosphorylation and phosphotransfer capability in vivo.     

An archaeal photosignal-transducing module mediates phototaxis in Escherichia coli

Halophilic archaea, such as Halobacterium salinarum and Natronobacterium pharaonis, alter their swimming behavior by phototaxis responses to changes in light intensity and color using visual pigment-like sensory rhodopsins (SRs). In N. pharaonis, SRII (NpSRII) mediates photorepellent responses through its transducer protein, NpHtrII. Here we report the expression of fusions of NpSRII and NpHtrII and fusion hybrids with eubacterial cytoplasmic domains and analyze their function in vivo in haloarchaea and in eubacteria. A fusion in which the C terminus of NpSRII is connected by a short flexible linker to NpHtrII is active in phototaxis signaling for H. salinarum, showing that the fusion does not inhibit functional receptor-transducer interactions. We replaced the cytoplasmic portions of this fusion protein with the cytoplasmic domains of Tar and Tsr, chemotaxis transducers from enteric eubacteria. Purification of the fusion protein from H. salinarum and Tar fusion chimera from Escherichia coli membranes shows that the proteins are not cleaved and exhibit absorption spectra characteristic of wild-type membranes. Their photochemical reaction cycles in H. salinarum and E. coli membranes, respectively, are similar to those of native NpSRII in N. pharaonis. These fusion chimeras mediate retinal-dependent phototaxis responses by Escherichia coli, establishing that the nine-helix membrane portion of the receptor-transducer complex is a modular functional unit able to signal in heterologous membranes. This result confirms a current model for SR-Htr signal transduction in which the Htr transducers are proposed to interact physically and functionally with their cognate sensory rhodopsins via helix-helix contacts between their transmembrane segments.     

Mechanism of CheA protein kinase activation in receptor signaling complexes.

The chemotaxis receptor for aspartate, Tar, generates responses by regulating the activity of an associated histidine kinase, CheA. Tar is composed of an extracellular sensory domain connected by a transmembrane sequence to a cytoplasmic signaling domain. The cytoplasmic domain fused to a leucine zipper dimerization domain forms soluble active ternary complexes with CheA and an adapter protein, CheW. The kinetics of kinase activity within these complexes compared to CheA alone indicate approximately a 50% decrease in the KM for ATP and a 100-fold increase in the Vmax. A truncated CheA construct that lacks the phosphoaccepting H-domain and the CheY/CheB-binding domain forms an activated ternary complex that is similar to the one formed by the full-length CheA protein. The Vmax of H-domain phosphorylation by this complex is enhanced approximately 60-fold, the KM for ATP decreased to 50%, and the KM for H-domain decreased to 20% of the values obtained with the same CheA construct in the absence of receptor and CheW. The kinetic data support a mechanism of CheA regulation that involves perturbation of an equilibrium between an inactive form where the H-domain is loosely bound and an active form where the H-domain is tightly associated with the CheA active site and properly positioned for phosphotransfer. The data are consistent with an asymmetric mechanism of CheA activation [Levit, M., Liu, I., Surette, M. G., and Stock, J. B. (1996) J. Biol. Chem. 271, 32057-32063] wherein only one phosphoaccepting domain of CheA at a time can interact with an active center within a CheA dimer.     

Solution structure of the bacterial chemotaxis adaptor protein CheW from Escherichia coli

The bacterial chemotaxis adaptor protein CheW physically links the chemoreceptors (MCPs) and the histidine kinase CheA. Extensive investigations using bacterium Escherichia coli have established the central role of CheW in the MCP-modulated activation of CheA. Here we report the solution structure of CheW from E. coli determined by NMR spectroscopy. The results show that E. coli CheW shares an overall fold with previously reported structure of CheW from Thermotoga maritima, whereas local conformational deviations are observed. In particular, the C-terminal alpha-helix is considerably longer in E. coli CheW and appears to shrink the active binding pocket with CheA. Our study provides the structural basis for further investigations in E. coli chemotaxis.     

Constitutively signaling fragments of Tsr, the Escherichia coli serine chemoreceptor.

Tsr, the serine chemoreceptor of Escherichia coli, has two signaling modes. One augments clockwise (CW) flagellar rotation, and the other augments counterclockwise (CCW) rotation. To identify the portion of the Tsr molecule responsible for these activities, we isolated soluble fragments of the Tsr cytoplasmic domain that could alter the flagellar rotation patterns of unstimulated wild-type cells. Residues 290 to 470 from wild-type Tsr generated a CW signal, whereas the same fragment with a single amino acid replacement (alanine 413 to valine) produced a CCW signal. The soluble components of the chemotaxis phosphorelay system needed for expression of these Tsr fragment signals were identified by epistasis analysis. Like full-length receptors, the fragments appeared to generate signals through interactions with the CheA autokinase and the CheW coupling factor. CheA was required for both signaling activities, whereas CheW was needed only for CW signaling. Purified Tsr fragments were also examined for effects on CheA autophosphorylation activity in vitro. Consistent with the in vivo findings, the CW fragment stimulated CheA, whereas the CCW fragment inhibited CheA. CheW was required for stimulation but not for inhibition. These findings demonstrate that a 180-residue segment of the Tsr cytoplasmic domain can produce two active signals. The CCW signal involves a direct contact between the receptor and the CheA kinase, whereas the CW signal requires participation of CheW as well. The correlation between the in vitro effects of Tsr signaling fragments on CheA activity and their in vivo behavioral effects lends convincing support to the phosphorelay model of chemotactic signaling.     

Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.     

CheW binding interactions with CheA and Tar. Importance for chemotaxis signaling in Escherichia coli

The initial signaling events underlying the chemotactic response of Escherichia coli to aspartic acid occur within a ternary complex that includes Tar (an aspartate receptor), CheA (a protein kinase), and CheW. Because CheW can bind to CheA and to Tar, it is thought to serve as an adapter protein in this complex. The functional importance of CheW binding interactions, however, has not been investigated. To better define the role of CheW and its binding interactions, we performed biochemical characterization of six mutant variants of CheW. We examined the ability of the purified mutant CheW proteins to bind to CheA and Tar, to promote formation of active ternary complexes, and to support chemotaxis in vivo. Our results indicate that mutations which eliminate CheW binding to Tar (V36M) or to CheA (G57D) result in a complete inability to form active ternary complexes in vitro and render the CheW protein incapable of mediating chemotaxis in vivo. The in vivo signaling pathway can, however, tolerate moderate changes in CheW-Tar and CheW-CheA affinities observed with several of the mutants (G133E, G41D, and 154ocr). One mutant (R62H) provided surprising results that may indicate a role for CheW in addition to binding CheA/receptors and promoting ternary complex formation.     

Chemotactic signaling by an Escherichia coli CheA mutant that lacks the binding domain for phosphoacceptor partners

CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheADeltaP2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheADeltaP2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheADeltaP2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheADeltaP2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.     

Template-directed assembly of receptor signaling complexes

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF. The extent of CheA activation was found to be independent of the level of covalent modification on the CF. Instead, the stability of the complex increased significantly as the level of covalent modification increased. Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR. Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.     

Structural analysis of bacterial chemotaxis proteins: components of a dynamic signaling system

Most motile bacteria are capable of directing their movement in response to chemical gradients, a behavior known as chemotaxis. The signal transduction system that mediates chemotaxis in enteric bacteria consists of a set of six cytoplasmic proteins that couple stimuli sensed by a family of transmembrane receptors to behavioral responses generated by the flagellar motors. Signal transduction occurs via a phosphotransfer pathway involving a histidine protein kinase, CheA, and a response regulator protein, CheY, that in its phosphorylated state, modulates the direction of flagellar rotation. Two auxiliary proteins, CheW and CheZ, and two receptor modification enzymes, methylesterase CheB and methyltransferase CheR, influence the flux of phosphoryl groups within this central pathway. This paper focuses on structural characteristics of the four signaling proteins (CheA, CheY, CheB, and CheR) for which NMR or x-ray crystal structures have been determined. The proteins are examined with respect to their signaling activities that involve reversible protein modifications and transient assembly of macromolecular complexes. A variety of data suggest conformational flexibility of these proteins, a feature consistent with their multiple roles in a dynamic signaling pathway.     

The cytoplasmic membrane-proximal domain of the HtrII transducer interacts with the E-F loop of photoactivated Natronomonas pharaonis sensory rhodopsin II

The structures of the cytoplasmic loops of the phototaxis receptor sensory rhodopsin II (SRII) and the membrane-proximal cytoplasmic domain of its bound transducer HtrII were examined in the dark and in the light-activated state by fluorescent probes and cysteine cross-linking. Light decreased the accessibility of E-F loop position 154 in the SRII-HtrII complex, but not in free SRII, consistent with HtrII proximity, which was confirmed by tryptophans placed within a 5-residue region identified in the HtrII membrane-proximal domain that exhibited Forster resonance energy transfer to a fluorescent probe at position 154 in SRII. The Forster resonance energy transfer was eliminated in the signaling deficient HtrII mutant G83F without loss of affinity for SRII. Finally, the presence of SRII and HtrII reciprocally inhibit homodimer disulfide cross-linking reactions in their membrane-proximal domains, showing that each interferes with the others self-interaction in this region. The results demonstrate close proximity between SRII-HtrII in the membrane-proximal domain, and in addition, light stimulation of the SRII inhibition of HtrII cross-linking was observed, indicating that the contact is enhanced in the photoactivated complex. A mechanism is proposed in which photoactivation alters the SRII-HtrII interaction in the membrane-proximal region during the signal relay process.     

Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of Natronomonas pharaonis

Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.     

Subunit organization in a soluble complex of tar,CheW, and CheA by electron microscopy

The Salmonella and Escherichia coli aspartate receptor, Tar, is representative of a large class of membrane receptors that generate chemotaxis responses by regulating the activity of an associated histidine protein kinase, CheA. Tar is composed of an NH(2)-terminal periplasmic ligand-binding domain linked through a transmembrane sequence to a COOH-terminal coiled-coil signaling domain in the cytoplasm. The isolated cytoplasmic domain of Tar fused to a leucine zipper sequence forms a soluble complex with CheA and the Src homology 3-like kinase activator, CheW. Activity of the CheA kinase in the soluble complex is essentially the same as in fully active complexes with the intact receptor in the membrane. The soluble complex is composed of approximately 28 receptor cytoplasmic domain chains, 6 CheW chains, and 4 CheA chains. It has a molecular weight of 1,400,000 (Liu, I., Levit, M., Lurz, R., Surette, M.G., and Stock, J.B. (1997) EMBO J. 16, 7231-7240). Electron microscopy reveals an elongated barrel-like structure with a largely hollow center. Immunoelectron microscopy has provided a general picture of the subunit and domain organization of the complex. CheA and CheW appear to be in the middle of the complex with the leucine zippers of the receptor construct at the ends. These findings show that the receptor signaling complex forms higher ordered structures with defined geometric architectures. Coupled with atomic models of the subunits, our results provide insights into the functional architecture by which the receptor regulates CheA kinase activity during bacterial chemotaxis.     

Adaptation mechanism of the aspartate receptor: electrostatics of the adaptation subdomain play a key role in modulating kinase activity

The aspartate receptor of the Escherichia coli and Salmonella typhimurium chemotaxis pathway generates a transmembrane signal that regulates the activity of the cytoplasmic kinase CheA. Previous studies have identified a region of the cytoplasmic domain that is critical to receptor adaptation and kinase regulation. This region, termed the adaptation subdomain, contains a high density of acidic residues, including specific glutamate residues that serve as receptor adaptation sites. However, the mechanism of signal propagation through this region remains poorly understood. This study uses site-directed mutagenesis to neutralize each acidic residue within the subdomain to probe the hypothesis that electrostatics in this region play a significant role in the mechanism of kinase activation and modulation. Each point mutant was tested for its ability to regulate chemotaxis in vivo and kinase activity in vitro. Four point mutants (D273N, E281Q, D288N, and E477Q) were found to superactivate the kinase relative to the wild-type receptor, and all four of these kinase-activating substitutions are located along the same intersubunit interface as the adaptation sites. These activating substitutions retained the wild-type ability of the attractant-occupied receptor to inhibit kinase activity. When combined in a quadruple mutant (D273N/E281Q/D288N/E477Q), the four charge-neutralizing substitutions locked the receptor in a kinase-superactivating state that could not be fully inactivated by the attractant. Similar lock-on character was observed for a charge reversal substitution, D273R. Together, these results implicate the electrostatic interactions at the intersubunit interface as a major player in signal transduction and kinase regulation. The negative charge in this region destabilizes the local structure in a way that enhances conformational dynamics, as detected by disulfide trapping, and this effect is reversed by charge neutralization of the adaptation sites. Finally, two substitutions (E308Q and E463Q) preserved normal kinase activation in vitro but blocked cellular chemotaxis in vivo, suggesting that these sites lie within the docking site of an adaptation enzyme, CheR or CheB. Overall, this study highlights the importance of electrostatics in signal transduction and regulation of kinase activity by the cytoplasmic domain of the aspartate receptor.     

Stimulus response coupling in bacterial chemotaxis: receptor dimers in signalling arrays

In the Escherichia coli chemotaxis system, a family of chemoreceptors in the cytoplasmic membrane binds stimulatory ligands and regulates the activity of an associated histidine kinase CheA to modulate swimming behaviour and thereby cause a net migration towards attractants and away from repellents. The chemoreceptors themselves have been shown to be predominantly dimeric, but in the presence of the kinase CheA plus an adapter protein, CheW, much higher order structures have been observed. Recent results indicate that transmembrane signalling occurs within receptor clusters rather than through isolated dimers. We propose that the mechanism involves receptor arrays where binding of ligands at the outside surface of the membrane affects lateral packing interactions that cause perturbations in the organization of the signalling array at the opposing surface of the membrane. Results with receptor chimeras as well as findings with tyrosine kinase receptors suggest that this mechanism may represent a common theme in membrane receptor function.