Control of chemotactic signal gain via modulation of a pre-formed receptor array.

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.

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

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 rod-shaped 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.