Two-component signaling pathways, a signal transduction motif that is widespread in prokaryotes and found in some eukaryotes, involve the autophosphorylation of histidine kinases and phosphotransfer to aspartyl groups of response regulators (1). Bacterial chemotaxis is a well-studied two-component signaling system that enables bacteria to sense chemical gradients and bias swimming toward larger attractant concentrations (2). The methyl-accepting chemotaxis receptors that provide the sensory input for this system have been the focus of numerous investigations that seek to understand the mechanism of transmembrane signaling. An intriguing property of these receptors is the formation of large receptor clusters, typically at the poles of the cell (3). These clusters are thought to be important for receptor cooperativity (4) and adaptation (5) to mediate the sensitivity, dynamic range, and integration of the chemotaxis signaling network (6–9). Clustering may also play a role in receptor activation, as observed for the EGF receptor and other receptors that are activated by ligand-induced dimerization or oligomerization (10, 11). Chemoreceptor clustering has been reported to vary with signaling state, but the evidence has been inconsistent. These data raise the question of whether a clustering equilibrium plays a role in the primary signal, ligand regulation of chemoreceptor activation of the kinase CheA. Four chemotaxis receptors in E coli share the overall structure and interaction sites shown in Figure 1 (12). The high abundance receptors Tar and Tsr detect aspartate and serine, respectively, and the low abundance receptors Trg and Tap detect ribose/galactose and dipeptides, respectively. These chemoreceptors are transmembrane alpha-helical homodimers, based on crystal and NMR structures of the periplasmic, HAMP, and cytoplasmic domains (13–15). The membrane-distal tip of the cytoplasmic domain binds two proteins, the histidine kinase CheA and a scaffolding protein CheW. The central region of the receptor cytoplasmic domain contains 4 glutamate residues that are methylated and demethylated, which enables the receptor to adapt to an ongoing stimulus. Both the CheR methyltransferase and the CheB methylesterase that modify these adaptation sites have been shown to bind to the carboxy terminus of the high abundance receptors (16, 17). Replacing Glu with Gln at the adaptation sites mimics the effects of receptor methylation (18), and the wild type receptor is genetically encoded in an intermediate adaptation state, with two Glu and two Gln (Gln are initially deamidated to Glu by CheB). Figure 1 The E. coli chemotaxis pathway depicted as a two state signaling system. Receptor/CheW/CheA complexes are shown in kinase-stimulating (green/magenta/green) and kinase-inhibiting (red/magenta/gray) states. The binding of attractant (filled triangles) inhibits ... Although not shown in Figure 1 for simplicity, chemotaxis receptors are interconnected by CheA and CheW into hexagonal arrays that have been observed across a wide range of prokaryotes (19). Furthermore, although the stoichiometry of the proteins in these arrays is not known, recent estimates suggest they contain more receptor than CheA and CheW: based on measured stoichiometries of six Tsr per CheA and CheW, it has been suggested that a pair of receptor trimer-of-dimers is needed to activate the dimeric CheA kinase (20). Recent cryoelectron tomography studies have yielded models for the arrangement of receptor, CheA and CheW in the signaling arrays (21, 22). Receptor signaling is usually described in terms of a two-state equilibrium, with attractant occupancy and methylation shifting the equilibrium in opposite directions (Figure 1). In the absence of attractant ligand, the receptor stimulates autophosphorylation of the histidine kinase CheA, which transfers the phosphate to either CheY or CheB. Phospho-CheY binds to the flagellar motor, causing a change from counterclockwise rotation of the flagellar bundle that propels the cell forward, to clockwise rotation that disrupts the flagellar bundle and causes the cell to tumble. Phosphorylation also activates the CheB methylesterase, which decreases the steady state level of receptor methylation that is determined by the relative activities of CheR and CheB. Binding of an attractant ligand to the receptor turns off kinase activation and thus decreases levels of phospho-CheY and tumbling frequency, so that the cell makes longer runs in the presence of attractants such as Asp or Ser. Following this rapid change in tumbling frequency, adaptation occurs on a slower timescale: decreased levels of phospho-CheB lead to increased levels of receptor methylation, shifting the equilibrium back towards the kinase-activating state. The attractant-bound receptor is also more efficiently methylated by CheR, which contributes to the adaptation shift back to the kinase-activating state. The focus of this study is to determine whether the extent of receptor clustering is different in the two signaling states depicted in Figure 1. We use the term cluster to refer to a receptor associated with other receptors, CheA, and CheW into an oligomeric multi-protein complex, with intermolecular contacts among the proteins within the cluster, as distinct from co-localized receptors that lack such contacts. Evidence from microscopy and in vivo cross-linking studies is mixed: some studies have reported that ligand binding decreases receptor clustering or methylation increases receptor clustering in cells. Libermann et al (23) observed an increase in the polar localization of CheA-containing complexes with methylation (Tar2Q2E ≈ half methylated receptor relative to Tar4E = unmethylated adaptation state), using fluorescence microscopy of E. coli expressing CheA fused to YFP (yellow fluorescent protein). However, because these changes were much smaller than the measured changes in kinase activity, they concluded that changes in assembly of CheA-containing clusters do not control the kinase (23). Based on immunoelectron microscopy of E. coli cells expressing a single type of chemotaxis receptor, Lyberger et al (24) reported that high abundance receptors are clustered independent of methylation state, but low abundance receptors are significantly less clustered in the unmethylated state. They suggested that both increases in abundance and methylation may shift the equilibrium toward a clustered state, and that such an equilibrium could also regulate kinase activation (24). Homma at al reported that attractant ligand does not decrease the polar localization of a Tar-GFP construct in E. coli, but attractant does decrease in vivo interdimer crosslinking of Tar (25). By contrast, Lamanna et al. observed that attractant ligand decreases polar clusters in both E. coli and B. subtilis, when receptors are crosslinked with paraformaldehyde and then visualized with a fluorescent antibody (26). Finally, Studdert & Parkinson reported that in vivo interdimer crosslinking of Tsr and Tar is independent of both ligand binding and methylation state (27). Limitations of these studies include the inability of microscopy to distinguish clustering (oligomerization) of receptors from co-localization, and the inability of crosslinking studies to distinguish whether changes in the extent of crosslinking result from conformational changes or dissociation of receptors. Two in vitro studies that correlated receptor concentration with changes in kinase activity are more suggestive that a clustering equilibrium may control the kinase. Lai et al. (28) varied the overexpression level of Tsr or Tar and isolated inner membrane vesicles that contained each receptor as a variable fraction of total membrane protein. The kinase activity per receptor increased linearly with receptor fraction, up to 50% of total membrane protein in these samples (28). Besschetnova et al (29) examined simpler, more defined samples of histidine-tagged cytoplasmic fragments of Tar4E, which were assembled on the surface of liposomes with Ni-chelating lipids, along with CheA and CheW. These template-assembled receptor-signalling arrays displayed a cooperative increase in kinase activation as the 2-dimensional receptor concentration on the vesicle increased. Moreover, receptor methylation activity was observed to decrease as the two dimensional concentration (density) of receptors increased, in a manner consistent with the signaling equilibrium of Figure 1 (29). The results of these in vitro studies are consistent with a clustering equilibrium model in which high receptor concentrations favor the kinase-activating state, which would be a larger oligomeric (more clustered) state than the methylation-activating state. Such a model predicts that ligand binding would favor receptor dissociation into the kinase-inactivating state (less clustered) and thus ligand affinity (which was not measured in either study) would also vary with receptor concentration. To test whether ligand-induced unclustering is an essential element of the mechanism of kinase regulation, we measured kinase activity and serine dose-response curves on purified E. coli Tsr reconstituted into liposomes over a range of two-dimensional concentrations of receptors. Our results indicate that the activity equilibrium does not involve a change in receptor oligomerization state. In combination with the previous template-assembly results (29), this indicates that the cytoplasmic domain of the kinase-off state has an expanded conformation.