title: Transmembrane signaling by the aspartate receptor: engineered disulfides reveal static regions of the subunit interface.
abstract: Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18-75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Chervitz
authors.forename: C M
authors.initials: CM
authors.lastname: Lin
authors.forename: J J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017459:Receptors, Amino Acid / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D012486:Salmonella typhimurium / Q000737:chemistry; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D004220:Disulfides; D017459:Receptors, Amino Acid; D011994:Recombinant Proteins; C031959:aspartic acid receptor; D003545:Cysteine