Chemotactic Signaling by Single-Chain Chemoreceptors

Abstract

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.

Engineered chemotaxis core signaling units indicate a constrained kinase-off state

“…been employed to modulate CheA activity Mowery, P, et al, 2015). Soluble chimeric receptors fragments containing a non-native trimerization motif pre-form a TOD arrangement and associate with CheA and CheW .…”

Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM

“…The realization that DosM spans the cytoplasmic complex is in contrast to a recent hypothesis based on the analysis of a singlechain Tsr chemoreceptor construct. This modified E. coli receptor contains a double signaling domain, and the results suggest that in this case, the two signaling domains directly interact with each other (29). We speculate that this structural difference is the result of the transmembrane domain in the Tsr construct that forces the signaling domains to interact with one another, whereas the lack of transmembrane domain in DosM prevents direct interaction of the two signaling domains favoring the linear architecture shown in the tomograms.…”

Design of a Histidine Kinase FRET Sensor to Detect Complex Signal Integration within Living Bacteria

“…Moreover, HKs can display cell-to-cell and subcellular activity variation. , One approach to track HK biochemical processes with subcellular resolution is fluorescence resonance energy transfer (FRET). FRET has been applied in vivo to track interactions between kinase and the response regulator, chemotaxis pathway protein interactions, − HK oligomerization, and HK heterodimerization …”