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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 rodshaped 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.S ensing and responding to extracellular signals are essential for any living cell. One of the most extensively studied sensing systems is the chemotaxis of Escherichia coli (1-4); i.e., migration toward or away from chemicals. The chemotactic signal transduction involves a His-Asp phosphate relay from the histidine kinase CheA to the response regulator CheY that is regulated by chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). The aspartate chemoreceptor Tar has a very low threshold concentration (Ϸ3 ϫ 10 Ϫ8 M) of L-aspartate for an attractant response (5-7). Moreover, E. coli responds to a very small change (Ͻ1%) in the receptor occupancy with aspartate (8). Therefore, an input signal has to be amplified to produce a significant response. The flagellar motor switching by phospho-CheY is a highly cooperative event that can account for at least some degree of signal amplification (9, 10). In addition, recent analyses by using fluorescence resonance energy transfer (11) suggested that much of the gain occurs at the receptor end of the signaling pathway. However, its mechanism remains to be elucidated: the gain could be achieved through the MCP-MCP interaction (12, 13) or the involvement of CheB (11,14).MCP forms a homodimer, regardless of its ligand occupancy state (15). Each subunit (Ϸ60 kDa) consists of two transmembrane helices (TM1 and TM2), the ligand-binding domain in the periplasm, the signaling͞adaptation domain in the cytoplasm, and the HAMP domain, which connects TM2 with the signaling͞ a...

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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.Chemotaxis of Escherichia coli is one of the most extensively studied sensory systems, recognizing the concentration of environmental chemicals and migrating toward the favorite direction (for reviews, see Refs. 1-5). All of the components have been identified and extensively studied. However, the molecular mechanisms underlying its high sensitivity and wide dynamic range have not been fully understood. The chemotactic signal is transmitted from the chemoreceptors to the flagellar motor via a stoichiometric His-Asp phosphorelay from the histidine kinase CheA to the response regulator CheY. The chemoreceptors of E. coli belong to one of the best studied transmembrane receptor families. The receptor cytoplasmic domain interacts with CheA and the adaptor protein CheW (6, 7), and the resulting ternary complexes form a cluster at a cell pole (8 -10). Attractant binding to the Tar dimer, which is formed regardless of its ligand occupancy state (11), induces a small but critical inward displacement of a membrane-spanning ␣-helix of one subunit (12-17). This displacement is thought to trigger a structural change in the cytoplasmic domain, which then inactivates CheA. To account for high sensitivity of the chemotaxis system, however, it has been proposed that attractant binding also affects neighboring receptor dimer(s) (18 -21), models that have been supported by several lines of evidence (21-26). Receptor clustering has also been implicated in signal gain control by methylation (or amidation) of specific glutamate residues that is responsible for adaptation to persisting stimuli. A slight decrease in the attractant binding affinity (27-30) and a slight increase in the CheA activity (28, 30) that result from receptor covalent modification cannot ac...