Crosslinking Snapshots of Bacterial Chemoreceptor Squads

The team signaling model for bacterial chemoreceptors proposes that receptor dimers of different detection specificities form mixed trimers of dimers. These receptor "squads" then recruit the cytoplasmic signaling proteins CheA and CheW to form ternary signaling teams, which typically cluster at the poles of the cell. We devised cysteine-directed in vivo crosslinking approaches to ask whether mixed receptor squads could form in the absence of CheA and CheW and, if so, whether the underlying structural interactions conformed to trimer-of-dimers geometry. One approach used cysteine reporters at positions in the serine (Tsr) and aspartate (Tar) receptors that should form disulfide-linked Tsr approximately Tar products when juxtaposed at the interface of a mixed trimer. Another approach used a cysteine reporter with trigonal geometry near the trimer contact region and a trifunctional maleimide reagent with a spacer length appropriate for capturing the three axial subunits in a trimer of dimers. Both approaches detected mixed receptor-crosslinking products in cells lacking CheA and CheW. Under these conditions, receptor methylation and ligand-binding state had no discernable effect on crosslinking efficiencies. Crosslinking with the trigonal reporter was rapid and did not increase with longer treatment times or higher reagent concentrations, suggesting that this method produces a short-exposure snapshot of the receptor population. The extent of crosslinking indicated that most of the cell's receptor molecules were organized in higher-order groups. Crosslinking in receptor trimer contact mutants correlated with their signaling behaviors, suggesting that trimers of dimers are both structural and functional precursors of chemoreceptor signaling teams in bacteria.