title: Mapping out regions on the surface of the aspartate receptor that are essential for kinase activation.
abstract: The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.
authors.affiliation: Department of Chemistry & Biochemistry and Molecular Biophysics Program, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Mehan
authors.forename: Noah C
authors.initials: NC
authors.lastname: White
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D001224:Aspartic Acid / Q000378:metabolism* / Q000502:physiology; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D005033:Ethylmaleimide / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D025941:Protein Interaction Mapping / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D017459:Receptors, Amino Acid / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D012235:Rhodamines / Q000737:chemistry; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D013499:Surface Properties
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D019151:Peptide Library; D017459:Receptors, Amino Acid; D012235:Rhodamines; C031959:aspartic acid receptor; D001224:Aspartic Acid; C005358:tetramethylrhodamine; C037041:fluorescein 5-maleimide; D011494:Protein Kinases; D003545:Cysteine; D005033:Ethylmaleimide