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

Abstract

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...

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

“…Previously reported in vivo cross-linking studies are consistent with the clusters of dimers that are seen in the lzTar C WA complexes (16,17), and EM analyses of cell sections have shown evidence of membrane folds at the cell poles that could support the type of receptor signaling domain-to-signaling domain juxtaposition seen in our soluble receptor͞signaling complexes (32). Given that the sequences of the receptor cytoplasmic domains, CheA, and CheW are highly conserved in virtually all chemotactic prokaryotes, it seems that this organization of the receptor complexes, a tight complex with two sets of signaling domains oriented toward one another, could reflect the structure of active receptor͞CheW͞CheA complexes in vivo.…”

“…Crosslinking studies of Tar and Tsr in membranes are consistent with a dimeric organization, although recent results indicate that higher order assemblies are probably involved (14)(15)(16)(17). Immuno-EM, immunofluorescence, and GFP-tagging studies have established that, within a cell, receptors, CheA, and CheW cluster together into large complexes that may contain thousands of subunits of each protein (18,19).…”

Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties

“…This effective signaling indicated that the fundamental conformational change of ligand-induced transmembrane signaling required no greater structural complexity than the homodimer. This conclusion implies that changes in trimer or higher order interactions that have been observed or postu- lated to be related to signaling (21)(22)(23)(24) are not necessary for coupling of ligand occupancy in the periplasmic domain to conformational change in the cytoplasmic domain. The notion of transmembrane signaling occurring within the structure of single dimers is consistent with the large body of data that identifies the conformational change of transmembrane signaling in the periplasmic and transmembrane domains as the piston sliding of a single signaling helix of a ligand-occupied receptor dimer (3,25).…”

Crosslinking snapshots of bacterial chemoreceptor squads

“…Kim et al (27) have proposed a model of the receptor network in which the periplasmic domains of neighboring trimerbased receptor-signaling teams contact one another. Those interactions could account for the crosslinking results of Homma et al (26).…”

“…Homma et al (26) recently described a crosslinking study of Tar with a cysteine reporter in the periplasmic, ligand-binding domain. They observed Tar oligomers consistent with a trimerof-dimers interpretation, but their crosslinking signal substantially depended on the presence of CheA and CheW and was significantly reduced on ligand binding.…”