Functional and structural effects of seven‐residue deletions on the coiled‐coil cytoplasmic domain of a chemoreceptor

Abstract

SummaryChemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiledcoil four-helix bundle. Changes in this domain during evolution are characterized by the presence of sevenresidue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces nonfunctional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.

A chemoreceptor from Pseudomonas putida forms active signalling complexes in Escherichia coli

“…We have previously demonstrated that Tar forms mixed trimers of dimers with two different 34H-class receptors from Rhodobacter sphaeroides, McpB and McpH, as well as with a Tsr-derived construct in which the deletion of a heptad in each arm of its cytoplasmic domain converts it into a 34H-like MCP (Massazza et al, 2012). In those experiments, mixed trimer formation was accompanied by a significant impairment of Tar-mediated signalling, suggesting that a structural distortion in the trimers interferes with function.…”

Bacterial chemoreceptors of different length classes signal independently

“…Variants carrying point mutations that suppressed the chemotaxis defects exhibited by the TsrH18 original construct were isolated as described in Massazza et al . ().…”

“…Candidate mutants were verified by sequencing the entire protein-coding region. Variants carrying point mutations that suppressed the chemotaxis defects exhibited by the TsrH18 original construct were isolated as described in Massazza et al (2012).…”