title: Hydrogen exchange differences between chemoreceptor signaling complexes localize to functionally important subdomains.
abstract: The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes.
authors.affiliation: Department of Chemistry, ‡Department of Biochemistry and Molecular Biology, and §Program in Molecular and Cellular Biology, University of Massachusetts , Amherst, Massachusetts 01003, United States.
authors.forename: Seena S
authors.initials: SS
authors.lastname: Koshy
authors.forename: Xuni
authors.initials: X
authors.lastname: Li
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Eyles
authors.forename: Robert M
authors.initials: RM
authors.lastname: Weis
authors.forename: Lynmarie K
authors.initials: LK
authors.lastname: Thompson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D007700:Kinetics; D008024:Ligands; D008081:Liposomes; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D009532:Nickel / Q000378:metabolism; D009829:Oleic Acids / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013386:Succinates / Q000737:chemistry; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins; C489103:CheW protein, E coli; D002614:Chelating Agents; D029968:Escherichia coli Proteins; D008024:Ligands; D008081:Liposomes; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C476561:N(alpha),N(alpha)-bis(carboxymethyl)-N(epsilon)-(1,2-dioleyl-sn-glycero-3-succinyl)-L-lysine; D009829:Oleic Acids; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D013386:Succinates; D009532:Nickel; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; C017251:1,2-oleoylphosphatidylcholine; D008239:Lysine