title: 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.
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.|Department of Biology, Hobart and William Smith Colleges, Geneva, New York, United States of America.
authors.forename: Patricia
authors.initials: P
authors.lastname: Mowery
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Ames
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.
authors.forename: Rebecca H
authors.initials: RH
authors.lastname: Reiser
authors.affiliation: Department of Biology, University of Utah, Salt Lake City, Utah, United States of America.
authors.forename: John S
authors.initials: JS
authors.lastname: Parkinson
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002633:Chemotaxis / Q000502:physiology*; D048168:Escherichia coli K12 / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607604:tsr protein, E coli; D011494:Protein Kinases; C091973:autophosphorylation-dependent multifunctional protein kinase; D000071677:Histidine Kinase; C000607608:cheA protein, E coli