CheA kinase and chemoreceptor interaction surfaces on CheW.
Chemotactic responses of Escherichia coli to aspartic acid are initiated by a ternary protein complex composed of Tar (chemoreceptor), CheA (kinase), and CheW (a coupling protein that binds to both Tar and CheA and links their activities). We used a genetic selection based on the yeast two-hybrid assay to identify nine cheW point mutations that specifically disrupted CheW interaction with CheA but not with Tar. We sequenced these single point mutants and purified four of the mutant CheW proteins for detailed biochemical characterizations that demonstrated the weakened affinity of the mutant CheW proteins for CheA, but not for Tar. In the three-dimensional structure of CheW, the positions affected by these mutations cluster on one face of the protein, defining a potential binding interface for interaction of CheW with CheA. We used a similar two-hybrid approach to identify four mutation sites that disrupted CheW binding to Tar. Mapping of these "Tar-sensitive" mutation sites and those from previous suppressor analysis onto the structure of CheW defined an extended surface on a face of the protein that is adjacent to the CheA-binding surface and that may serve as an interface for CheW binding to Tar.
Boukhvalova
Marina
M
Department of Cell Biology and Molecular Genetics and Graduate Program in Molecular and Cellular Biology, University of Maryland, College Park, Maryland 20742, USA.
VanBruggen
Ricaele
R
Stewart
Richard C
RC
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002628:Chemoreceptor Cells; D002633:Chemotaxis; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D000071677:Histidine Kinase; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D017354:Point Mutation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011956:Receptors, Cell Surface; C057331:Tar protein, E coli; C051236:CheW protein, Bacteria; D000071677:Histidine Kinase; C000607608:cheA protein, E coli