title: Engineered socket study of signaling through a four-helix bundle: evidence for a yin-yang mechanism in the kinase control module of the aspartate receptor.
abstract: The chemoreceptors of Escherichia coli and Salmonella typhimurium form stable oligomers that associate with the coupling protein CheW and the histidine kinase CheA to form an ultrasensitive, ultrastable signaling lattice. Attractant binding to the periplasmic domain of a given receptor dimer triggers a transmembrane conformational change transmitted through the receptor to its cytoplasmic kinase control module, a long four-helix bundle that binds and regulates CheA kinase. The kinase control module comprises three functional regions: the adaptation region possessing the receptor adaptation sites, a coupling region that transmits signals between other regions, and the protein interaction region possessing contact sites for receptor oligomerization and for CheA-CheW binding. On the basis of the spatial clustering of known signal locking Cys substitutions and engineered disulfide bonds, this study develops the yin-yang hypothesis for signal transmission through the kinase control module. This hypothesis proposes that signals are transmitted through the four-helix bundle via changes in helix-helix packing and that the helix packing changes in the adaptation and protein interaction regions are tightly and antisymmetrically coupled. Specifically, strong helix packing in the adaptation region stabilizes the receptor on state, while strong helix packing in the protein interaction region stabilizes the off state. To test the yin-yang hypothesis, conserved sockets likely to strengthen specific helix-helix contacts via knob-in-hole packing interactions were identified in the adaptation, coupling, and protein interaction regions. For 32 sockets, the knob side chain was truncated to Ala to weaken the knob-in-hole packing and thereby destabilize the local helix-helix interaction provided by that socket. We term this approach a "knob truncation scan". Of the 32 knob truncations, 28 yielded stable receptors. Functional analysis of the signaling state of these receptors revealed seven lock-off knob truncations, all located in the adaptation region, that trap the receptor in its "off" signaling state (low kinase activity, high methylation activity). Also revealed were five lock-on knob truncations, all located in the protein interaction region, that trap the "on" state (high kinase activity, low methylation activity). These findings provide strong evidence that a yin-yang coupling mechanism generates concerted, antisymmetric helix-helix packing changes within the adaptation and protein interaction regions during receptor on-off switching. Conserved sockets that stabilize local helix-helix interactions play a central role in this mechanism: in the on state, sockets are formed in the adaptation region and disrupted in the protein interaction region, while the opposite is true in the off state.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biophysics Program, University of Colorado, Boulder, Colorado 80309-0215, USA.
authors.forename: Kalin E
authors.initials: KE
authors.lastname: Swain
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Gonzalez
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Falke
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002633:Chemotaxis / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008780:Methyltransferases / Q000737:chemistry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D025941:Protein Interaction Mapping / Q000379:methods*; D017433:Protein Structure, Secondary / Q000235:genetics; D017459:Receptors, Amino Acid / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D017459:Receptors, Amino Acid; C031959:aspartic acid receptor; C069656:CheB protein, Bacteria; D001224:Aspartic Acid; C513923:CheR protein, E coli; D008780:Methyltransferases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli
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