Insights into the organization and dynamics of bacterial chemoreceptor clusters through in vivo crosslinking studies

Abstract

The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers that bind the cytoplasmic proteins CheA and CheW to form ternary signaling complexes clustered at the cell poles. We used a trifunctional crosslinking reagent targeted to cysteine residues in the aspartate (Tar) and serine (Tsr) receptors to obtain in vivo snapshots of trimer composition in the receptor population. To analyze the dynamics of trimer formation, we followed the appearance of mixed trimers when cells expressing Tar were induced for the expression of Tsr and treated with the crosslinker shortly after the onset of induction. In the absence of CheA or CheW, preformed Tar trimers exchanged partners readily with newly made Tsr. Conversely, in the presence of CheA and CheW, receptor trimers seldom exchanged partners, irrespective of the presence or absence of attractants. The C-terminal receptorcoupling domain of the CheA kinase, which contains binding determinants for the CheW protein, was essential for conferring low exchangeability to the preformed trimers of dimers. CheW also was required for this effect, but, unlike CheA, overexpression of CheW interfered with trimer formation and chemotactic behavior. The CheW effect probably occurs through binding interactions that mask the receptor sites needed for trimer formation. We propose that clustered receptors are organized in mixed trimers of dimers through binding interactions with CheA and CheW, which play distinctly different architectural roles. Moreover, once complete signaling teams have formed, they no longer undergo dynamic exchange of receptor members.chemotaxis ͉ epistasis ͉ receptor clustering ͉ signaling teams ͉ trimer of dimers E scherichia coli and other motile bacteria monitor their chemical environment with high sensitivity and broad detection ranges and use this information to seek out favorable living conditions. These chemotactic behaviors of bacteria offer tractable models for investigating the molecular basis of biological chemosensing and signal amplification. Indeed, considerable progress has been made in documenting the high-gain signaling properties of bacterial chemoreceptors, but their underlying molecular mechanisms remain elusive (recently reviewed in refs. 1 and 2).Methyl-accepting chemotaxis proteins (MCPs) are the predominant chemoreceptors in bacteria (3). E. coli possesses five MCP-like receptors with different detection specificities; its most abundant types are the serine receptor (Tsr) and the aspartate receptor (Tar) (4). MCPs are integral membrane proteins characterized by a conserved cytoplasmic domain that interacts with the coupling protein CheW and the histidine kinase CheA to form ternary signaling complexes, which communicate with the cell's flagellar motors through protein phosphorylation pathways (1, 2). Tar and Tsr have periplasmic sensing domains that monitor chemoeffector levels through high-affinity binding sites. Changes in ligand occupancy modulate MCP sig...

Bacterial chemoreceptors: high-performance signaling in networked arrays

“…However, trimers assembled in the absence of either CheA or CheW exchange members with recently made receptor molecules, whereas trimers formed in the presence of both CheA and CheW do not undergo such exchanges [29]. Thus, CheA and CheW stabilize trimer arrangements, most likely through binding interactions within and/or between trimer units.…”

“…The stoichiometries of receptor, CheA and CheW molecules during ternary complex assembly have profound effects on the resultant structure and function of the array. For example, an excess of CheW interferes with trimer formation, probably through binding interactions that mask the trimer contact surfaces on receptor molecules [29]. Similarly, CheW and CheA compete for binding sites on receptor molecules [30,31].…”

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

“…Although large changes in membrane architecture may not occur on this time scale, local movements could permit additional CheA and receptor proteins near the edge of an active receptor͞signaling complex to join into it and thus increase the net CheA activity. Additionally, conformational changes in the receptors seem to be able to inactivate CheA without requiring dissociation (24,29,38). The receptors outside the active receptor͞signaling complexes may still play an important role in cellular memory and adaptation, because these receptors would be substrates for attractant-mediated methylation and demethylation by CheR and CheB (39)(40)(41)(42).…”

Conformational suppression of inter-receptor signaling defects

“…1) and that these structural interactions underlie cooperative signaling between different types of receptors, which has been observed both in vivo (6,7) and in vitro (19). It seems unlikely that trimers represent a transitory step in cluster assembly because all of the cell's receptor molecules, not just those newly synthesized, exhibit crosslinking patterns consistent with trimer geometry (12,20). Moreover, the CheA and CheW proteins stabilize the members of receptor teams against exchanges with other trimers, implying that trimer-based interactions between different receptors persist while they are actively signaling (20).…”