Aspartate Chemoreceptor Research Articles

Observing transmembrane conformational signaling with smFRET.

Transmembrane receptors transmit information about the local chemical environment across the membrane to regulate cellular signaling. The conformational changes responsible for signal transduction have been inaccessible due to the challenges associated with isolating and interrogating full-length receptors. As a result, studies have been limited to individual domains, where the transmembrane conformational dynamics are inherently missing. We overcame these limitations by embedding full-length receptors within membrane nanodiscs, a lipid bilayer encircled by a solubilizing belting protein, and using single-molecule Förster resonance energy transfer (FRET) to follow the transmembrane conformational changes through the receptor. First, in the aspartate chemoreceptor from E. coli we find a change in the cytoplasmic helical packing and dynamics upon ligand occupancy, which may be a general mechanism in coiled-coil signaling proteins. Second, in the mammalian epidermal growth factor receptor (EGFR), we identify a ligand- and lipid-dependent conformational change of the intracellular domain upon extracellular ligand binding, revealing a previously unknown transmembrane conformational coupling that, remarkably, is mediated by a single transmembrane alpha-helix.

A peculiar case of Campylobacter jejuni attenuated aspartate chemosensory mutant, able to cause pathology and inflammation in avian and murine model animals

An attenuated Campylobacter jejuni aspartate chemoreceptor ccaA mutant caused gross pathological changes despite reduced colonisation ability in animal models. In chickens, the pathological changes included connective tissue and thickening of the mesenteric fat, as well as the disintegration of the villus tips in the large intestine, whereas in mice, hepatomegaly occurred between 48–72 hours post infection and persisted for the six days of the time course. In addition, there was a significant change in the levels of IL-12p70 in mice infected with the C. jejuni ccaA mutant. CcaA isogenic mutant was hyper-invasive in cell culture and microscopic examination revealed that it had a “run” bias in its “run-and-tumble” chemotactic behaviour. The mutant cells also exhibited lower level of binding to fucosylated and higher binding to sialylated glycan structures in glycan array analysis. This study highlights the importance of investigating phenotypic changes in C. jejuni, as we have shown that specific mutants can cause pathological changes in the host, despite reduction in colonisation potential.

Membrane Curvature and the Tol-Pal Complex Determine Polar Localization of the Chemoreceptor Tar in Escherichia coli.

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years, different mechanisms have been put forward to explain this polar localization, including stochastic clustering, membrane curvature-driven localization, interactions with the Tol-Pal complex, and nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at the cell poles. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterized by strong membrane curvature. Chemoreceptors, like Tar, form trimers of dimers that bend the cell membrane due to a rigid tripod structure. The curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favorable, as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or the cell poles. These findings favor a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex.IMPORTANCE Bacteria have exquisite mechanisms to sense and adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli and found that membrane curvature at cell division sites and the Tol-Pal protein complex localize Tar at cell division sites, the future cell poles. This study shows how membrane curvature can guide localization of proteins in a cell.

Tuning Chemoreceptor Signaling by Positioning Aromatic Residues at the Lipid-Aqueous Interface.

Aromatic tuning facilitates stimulus-independent modulation of receptor output. The methodology is based upon the affinity of amphipathic aromatic residues, namely Trp and Tyr, for the polar-hydrophobic interfaces found within biological membranes. Here, we describe the application of aromatic tuning within the aspartate chemoreceptor of Escherichia coli (Tar). We have also employed the method within other related proteins, such as sensor histidine kinases (SHKs), and therefore hope that other research groups find it useful to modulate signal output from their receptor of interest.

Employing aromatic tuning to modulate output from two-component signaling circuits.

Two-component signaling circuits (TCSs) govern the majority of environmental, pathogenic and industrial processes undertaken by bacteria. Therefore, controlling signal output from these circuits in a stimulus-independent manner is of central importance to synthetic microbiologists. Aromatic tuning, or repositioning the aromatic residues commonly found at the cytoplasmic end of the final TM helix has been shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor (EnvZ) of Escherichia coli. Aromatic residues are found in a similar location within other bacterial membrane-spanning receptors, suggesting that aromatic tuning could be harnessed for a wide-range of applications. Here, a brief synopsis of the data underpinning aromatic tuning, the initial successes with the method and the inherent advantages over those previously employed for modulating TCS signal output are presented.

Both piston-like and rotational motions are present in bacterial chemoreceptor signaling.

Bacterial chemotaxis signaling is triggered by binding of chemo-effectors to the membrane-bound chemoreceptor dimers. Though much is known about the structure of the chemoreceptors, details of the receptor dynamics and their effects on signaling are still unclear. Here, by using molecular dynamics simulations and principle component analysis, we study the dynamics of the periplasmic domain of aspartate chemoreceptor Tar dimer and its conformational changes when binding to different ligands (attractant, antagonist, and two attractant molecules). We found two dominant components (modes) in the receptor dynamics: a relative rotation of the two Tar monomers and a piston-like up-and-down sliding movement of the α4 helix. These two modes are highly correlated. Binding of one attractant molecule to the Tar dimer induced both significant piston-like downward movements of the α4 helix and strong relative rotations of the two Tar monomers, while binding of an antagonist or the symmetric binding of two attractant molecules to a Tar dimer suppresses both modes. The anti-symmetric effects of the relative rotation mode also explained the negative cooperativity between the two binding pockets. Our results suggest a mechanism of coupled rotation and piston-like motion for bacterial chemoreceptor signaling.

Differential repositioning of the second transmembrane helices from E. coli Tar and EnvZ upon moving the flanking aromatic residues

Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.

Multidimensional Solid-State NMR of Functional Chemotaxis Receptor Signaling Complexes

Bacterial chemotaxis receptors serve as an ideal model system for elucidating molecular mechanisms of transmembrane signaling, due to the experimental accessibility and established biochemical characterization of these proteins. A gap in the structural understanding of bacterial chemotaxis is present between electron microscopy of the intact arrays of receptor complexes found in cells and high-resolution crystal structures of individual proteins and truncated complexes. To bridge this gap we have initiated multidimensional solid-state NMR spectroscopy studies of a kinase-active ternary complex composed of the soluble 30 kDa cytoplasmic fragment (CF) of the aspartate chemoreceptor, the 70 kDa histidine kinase CheA, and the 18 kDa adaptor protein CheW. We prepared uniformly 13C & 15N labeled CF and obtained both homonuclear and heteronuclear correlation spectra of frozen and unfrozen complexes.The initial data indicates that such experiments are feasible on this functional multi-protein complex. A number of narrow resonances with sub-ppm linewidths are observed in spectra of both frozen and unfrozen samples, and both states exhibit sufficient signal-to-noise for acquiring multidimensional spectra. Overall, the spectra demonstrate the strongly overlapping resonances expected for an alpha-helical 30 kDa protein, which suggests selective labeling for future measurements of structural constraints. For these samples containing uniformly labeled receptor fragment, comparison with predicted chemical shift spectra are being used to test the current structural model. Comparisons of frozen and unfrozen spectra are also being used to assess the presence of static disorder or dynamics in the side chain and backbone resonances. The goal for uniformly labeled receptor samples is to compare spectral properties of both signaling states to gain insight into possible differences in structure and dynamics.This research supported by NIH grant GM085288.

Residues at the Cytoplasmic End of Transmembrane Helix 2 Determine the Signal Output of the TarEc Chemoreceptor

Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia coli aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild-type Tar(Ec), is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six-residue sequence at positions 207-212. Moving various combinations of aromatic residues among these positions generates substantial changes in receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.

3PT191 大腸菌走化性受容体Tarシステイン置換シリーズの化学架橋によるクラスター構造推定(日本生物物理学会第50回年会(2012年度))

3PT191 大腸菌走化性受容体Tarシステイン置換シリーズの化学架橋によるクラスター構造推定(日本生物物理学会第50回年会(2012年度))

3PT186 大腸菌アスパラギン酸走性受容体Tarによる忌避物質Ni^<2+>感知機構の解析(日本生物物理学会第50回年会(2012年度))

3PT186 大腸菌アスパラギン酸走性受容体Tarによる忌避物質Ni^<2+>感知機構の解析(日本生物物理学会第50回年会(2012年度))

Mutational Analysis of N381, a Key Trimer Contact Residue in Tsr, the Escherichia coli Serine Chemoreceptor

Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery.

Structural characterization of AS1–membrane interactions from a subset of HAMP domains

HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZEc, the E. coli nitrate/nitrite sensor NarXEc, and the aspartate chemoreceptor of E. coli (TarEc). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity within AS1 from NarXEc and TarEc but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from TarEc associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1–membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.

Comprehensive Analysis of HAMP Domains: Implications for Transmembrane Signal Transduction

Homodimeric receptors with one or two transmembrane (TM) segments per monomer are universal to life and represent the largest and most diverse group of cellular TM receptors. They frequently share domain types across phyla and, in some cases, have been recombined experimentally into functional chimeras (e.g., the bacterial aspartate chemoreceptor with the human insulin receptor), suggesting that they have a common mechanism. The nature of this mechanism, however, is still being debated. We have proposed a new model for transduction mechanism by axial helix rotation, based on the structure of a widespread domain, HAMP, that frequently occurs in direct continuation of the last TM segment, primarily in histidine kinases and chemoreceptors. Here we show by statistical analysis that HAMP domain sequences have biophysical properties compatible with the two conformations proposed by the model. The analysis also identifies three networks of coevolving residues, which allow the mechanism to subdivide into individual steps. The most extended of these networks is specific for membrane-bound HAMP domains and most likely accepts the signal from the TM helices. In a classification based on sequence clustering, these HAMPs form a central supercluster, surrounded by smaller clusters of divergent HAMPs, which typically combine into arrays of up to 31 consecutive copies and accept conformational input from other HAMP domains. Unexpectedly, the classification shows a division between domains of histidine kinases and those of chemoreceptors; thus, except for a few versatile lineages, HAMP domains are largely specific for one particular output domain. Within proteins using a given output domain, HAMP domains also show extensive coevolution with histidine kinases, but not with chemoreceptors. We attribute the greater capability for recombination among chemoreceptors to their acquisition of a reversible modification system, which acts as a capacitor for the initially deleterious effects of combining domains optimized in different contexts.

Mechanisms Underlying Subcellular Localization of the Bacterial Transmembrane Chemoreceptor

The chemoreceptors of Escherichia coli cluster at a cell pole, a property which is critical for signaling. However, little is known about the mechanism of polar localization. Our recent study demonstrated that the aspartate chemoreceptor (Tar)-GFP fusion protein is inserted into lateral membrane regions and migrates to the pole. Unexpectedly, Tar-GFP was found to be arranged into a coil, which reflects a coil of the Sec protein translocation machinery. The Sec coil appeared distinct from the coil of MreB, an actin-like cytoskeletal protein. These findings shed new light on the spatial organization of membrane proteins in E. coli.

1P-238 大腸菌アスパラギン酸受容体Tar HAMPドメインのシグナル伝達および細胞内局在における役割(化学受容,第46回日本生物物理学会年会)

1P-238 大腸菌アスパラギン酸受容体Tar HAMPドメインのシグナル伝達および細胞内局在における役割(化学受容,第46回日本生物物理学会年会)

Formation and Activity of Template-Assembled Receptor Signaling Complexes

Problems in membrane biology require methods to recreate the interactions between receptors and cytoplasmic signaling proteins at the membrane surface. Here, unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and a nickel-chelating lipid were used as templates to direct the assembly of proteins from the Escherichia coli chemotaxis signaling pathway. The bacterial chemoreceptors are known to form clusters, which promote the binding of the adaptor protein (CheW) and the kinase (CheA). When CheA was incubated with vesicles, CheW, and a histidine-tagged cytoplasmic domain fragment of the aspartate chemoreceptor (CF), the kinase activity was stimulated approximately 300-fold. Activity and pull-down assays were used with dynamic light scattering and electron microscopy to characterize the protein-vesicle compositions that were correlated with the high levels of activity, which demonstrated that CF-CheW-CheA complexes on the vesicle surface were the active entities. Assembly and stimulation occurred with vesicles of different sizes and CFs in different extents of glutamine substitution (in place of glutamate) at physiologically relevant sites. An exception was the combination of sonicated vesicles with the unsubstituted CF, which displayed lower CheA activity. The lower activity was attributed to the high curvature of the sonicated vesicles and a weaker tendency of the unsubstituted CF to self-assemble. Electron micrographs of the vesicle-protein assemblies revealed that protein binding induced pronounced changes in vesicle shape, which was consistent with the introduction of positive curvature in the outer leaflet of the bilayer. Overall, vesicle-mediated template-directed assembly is shown to be an effective way to form functional complexes of membrane-associated proteins and suggests that significant changes in membrane shape can be involved in the process of transmembrane signaling.

3P205 大腸菌アスパラギン酸受容体TarのHAMPドメイン内の1つのアミノ酸残基置換がそのクラスター形成と極局在に与える影響(細胞生物学的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

3P205 大腸菌アスパラギン酸受容体TarのHAMPドメイン内の1つのアミノ酸残基置換がそのクラスター形成と極局在に与える影響(細胞生物学的課題(接着・運動・骨格・伝達・膜),ポスター発表,第45回日本生物物理学会年会)

Tuning a Bacterial Chemoreceptor with Protein−Membrane Interactions

Chemoreceptors in Escherichia coli are homodimeric transmembrane proteins that convert environmental stimuli into intracellular signals controlling flagellar motion. Chemoeffectors bind to the extracellular (periplasmic) domain of the receptors, whereas their cytoplasmic domain mediates signaling and adaptation. The second transmembrane helix (TM2) connects these two domains. TM2 contains an aliphatic core flanked by amphipathic aromatic residues that have specific affinity for polar-hydrophobic membrane interfaces. We previously showed that Trp-209, near the cytoplasmic end of TM2, helps maintain the normal baseline-signaling state of the aspartate chemoreceptor (Tar) and that Tyr-210 plays an auxiliary role in this control. We have now repositioned the Trp-209/Tyr-210 pair in single-residue increments about the cytoplasmic polar-hydrophobic interface. Changes from WY-2 to WY+1 modulate the baseline-signaling state of the receptor in predictable and incremental steps that can be compensated by adaptive methylation/demethylation. Greater displacements, as in WY-3, WY+2, and WY+3, bias the receptor to the off kinase-inhibiting state or the on kinase-stimulating state, respectively, to a degree that cannot be fully compensated by the adaptation system. Aromatic residues analogous to Trp-209/Tyr-210 are present in other chemoreceptors and many transmembrane sensor kinases, where they may serve a similar function.

Control of Chemotactic Signal Gain via Modulation of a Pre-formed Receptor Array

The remarkably wide dynamic range of the chemotactic pathway of Escherichia coli, a model signal transduction system, is achieved by methylation/amidation of the transmembrane chemoreceptors that regulate the histidine kinase CheA in response to extracellular stimuli. The chemoreceptors cluster at a cell pole together with CheA and the adaptor CheW. Several lines of evidence have led to models that assume high cooperativity and sensitivity via collaboration of receptor dimers within a cluster. Here, using in vivo disulfide cross-linking assays, we have demonstrated a well defined arrangement of the aspartate chemoreceptor (Tar). The differential effects of amidation on cross-linking at different positions indicate that amidation alters the relative orientation of Tar dimers to each other (presumably inducing rotational displacements) without much affecting the conformation of the periplasmic domains. Interestingly, the effect of aspartate on cross-linking at any position tested was roughly opposite to that of receptor amidation. Furthermore, amidation attenuated the effects of aspartate by several orders of magnitude. These results suggest that receptor covalent modification controls signal gain by altering the arrangement or packing of receptor dimers in a pre-formed cluster.