title: Defining a key receptor-CheA kinase contact and elucidating its function in the membrane-bound bacterial chemosensory array: a disulfide mapping and TAM-IDS Study.
abstract: The three core components of the ubiquitous bacterial chemosensory array - the transmembrane chemoreceptor, the histidine kinase CheA, and the adaptor protein CheW - assemble to form a membrane-bound, hexagonal lattice in which receptor transmembrane signals regulate kinase activity. Both the regulatory domain of the kinase and the adaptor protein bind to overlapping sites on the cytoplasmic tip of the receptor (termed the protein interaction region). Notably, the kinase regulatory domain and the adaptor protein share the same fold constructed of two SH3-like domains. The present study focuses on the structural interface between the receptor and the kinase regulatory domain. Two models have been proposed for this interface: Model 1 is based on the crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheW adaptor protein. This model has been used in current models of chemosensory array architecture to build the receptor-CheA kinase interface. Model 2 is based on a newly determined crystal structure of a homologous Thermotoga complex between a receptor fragment and the CheA kinase regulatory domain. Both models present unique strengths and weaknesses, and current evidence is unable to resolve which model best describes contacts in the native chemosensory arrays of Escherichia coli, Salmonella typhimurium, and other bacteria. Here we employ disulfide mapping and tryptophan and alanine mutation to identify docking sites (TAM-IDS) to test Models 1 and 2 in well-characterized membrane-bound arrays formed from E. coli and S. typhimurium components. The results reveal that the native array interface between the receptor protein interaction region and the kinase regulatory domain is accurately described by Model 2, but not by Model 1. In addition, the results show that the interface possesses both a structural function that contributes to stable CheA kinase binding in the array and a regulatory function central to transmission of the activation signal from receptor to CheA kinase. On-off switching alters the disulfide formation rates of specific Cys pairs at the interface, but not most Cys pairs, indicating that signaling perturbs localized regions of the interface. The findings suggest a simple model for the rearrangement of the interface triggered by the attractant signal and for longer range transmission of the signal in the chemosensory array.
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biophysics Program, University of Colorado , Boulder, Colorado 80309-0215, United States.
authors.forename: Kene N
authors.initials: KN
authors.lastname: Piasta
authors.forename: Caleb J
authors.initials: CJ
authors.lastname: Ulliman
authors.forename: Peter F
authors.initials: PF
authors.lastname: Slivka
authors.forename: Brian R
authors.initials: BR
authors.lastname: Crane
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000409:Alanine / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry*; D002633:Chemotaxis / Q000502:physiology*; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000737:chemistry*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008956:Models, Chemical; D062105:Molecular Docking Simulation; D011494:Protein Kinases / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D014364:Tryptophan / Q000235:genetics
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D014364:Tryptophan; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D000409:Alanine