Effects of glutamines and glutamates at sites of covalent modification of a methyl-accepting transducer.
Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.
Park
C
C
Department of Biological Science/Engineering, Korea Advanced Institute of Science and Technology, Cheongyang, Seoul.
Dutton
D P
DP
Hazelbauer
G L
GL
D001426:Bacterial Proteins; D001665:Binding Sites; D002633:Chemotaxis; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000502:physiology*; D005971:Glutamates / Q000502:physiology; D005973:Glutamine / Q000502:physiology; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000502:physiology; D000072236:Methyl-Accepting Chemotaxis Proteins; D009154:Mutation; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D005971:Glutamates; D008024:Ligands; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D005973:Glutamine