In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate.
In Escherichia coli, the ammonia channel AmtB and the P(II) signal transduction protein GlnK constitute an ammonium sensory system that effectively couples the intracellular nitrogen regulation system to external changes in ammonium availability. Binding of GlnK to AmtB apparently inactivates the channel, thereby controlling ammonium influx in response to the intracellular nitrogen status. We designed an N-terminally histidine-tagged version of AmtB with a native C-terminal region in order to purify the AmtB-GlnK complex. Purification revealed a stable and direct interaction between AmtB and GlnK, thereby showing for the first time that stability of the complex does not require other proteins. The stoichiometry of the complex was determined by two independent approaches, both of which indicated a 1:1 ratio of AmtB to GlnK. We also showed by mass spectrometry that only the fully deuridylylated form of GlnK co-purifies with AmtB. The purified complex allowed in vitro studies of dissociation and association of AmtB and GlnK. The interaction of GlnK with AmtB is dependent on ATP and is also sensitive to 2-oxoglutarate. Our in vitro data suggest that in vivo association and dissociation of the complex might not only be dependent on the uridylylation status of GlnK but may also be influenced by intracellular pools of ATP and 2-oxoglutarate.
Durand
Anne
A
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom.
Merrick
Mike
M
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000502:physiology*; D027682:Cation Transport Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D051922:PII Nitrogen Regulatory Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding
C111096:AmtB protein, E coli; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D051922:PII Nitrogen Regulatory Proteins; D000255:Adenosine Triphosphate; C497878:glnK protein, E coli; D009713:Nucleotidyltransferases