Template-directed assembly of receptor signaling complexes.
Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup. In the presence of the adaptor protein CheW, CheA bound to and was activated approximately 180-fold by vesicle-bound CF. The extent of CheA activation was found to be independent of the level of covalent modification on the CF. Instead, the stability of the complex increased significantly as the level of covalent modification increased. Surface-assembled CF was also found to serve as a substrate for receptor methylation in a reaction catalyzed by the receptor methyltransferase, CheR. Since neither CheA activation nor CF methylation was observed in comparable samples in the absence of vesicles, it is concluded that surface templating generates the organization among CF subunits required for biochemical activity.
Shrout
Anthony L
AL
Department of Chemistry, LGRT 701, 710 North Pleasant Street, University of Massachusetts, Amherst, Massachusetts 01003-9336, USA.
Montefusco
David J
DJ
Weis
Robert M
RM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D002628:Chemoreceptor Cells; D002633:Chemotaxis / Q000502:physiology; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000071677:Histidine Kinase; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D008780:Methyltransferases / Q000378:metabolism; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D008055:Lipids; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; C051236:CheW protein, Bacteria; D008780:Methyltransferases; C038706:chemotaxis methyltransferase; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D000251:Adenosine Triphosphatases