The crystal structure of the Escherichia coli AmtB–GlnK complex reveals how GlnK regulates the ammonia channel

Abstract

Amt proteins are ubiquitous channels for the conduction of ammonia in archaea, eubacteria, fungi, and plants. In Escherichia coli, previous studies have indicated that binding of the P II signal transduction protein GlnK to the ammonia channel AmtB regulates the channel thereby controlling ammonium influx in response to the intracellular nitrogen status. Here, we describe the crystal structure of the complex between AmtB and GlnK at a resolution of 2.5 Å. This structure of P II in a complex with one of its targets reveals physiologically relevant conformations of both AmtB and GlnK. GlnK interacts with AmtB almost exclusively via a long surface loop containing Y51 (T-loop), the tip of which inserts deeply into the cytoplasmic pore exit, blocking ammonia conduction. Y51 of GlnK is also buried in the pore exit, explaining why uridylylation of this residue prevents complex formation.regulation ͉ x-ray structure ͉ PII protein

The crystal structure of the complex of P II and acetylglutamate kinase reveals how P II controls the storage of nitrogen as arginine

“…By similarity with M. jannaschii GlnK1, MgATP, and ADP binding to P II should trigger, respectively, the compact and highly extended T-loop conformations (13,15), explaining the dissociation of the P II -NAGK complex by ADP but not by MgATP (6). These MgATP and ADP effects on the T-loop fit the proposed P II role in energy signaling in cyanobacteria (10).…”

“…The indirect effects of P II on NAGK functionality contrast with the direct role of GlnK in blocking the ammonia channel AmtB (14,15). In the latter case, the tip of the ADP-binding and more extended T-loop, including nonuridylylated Y51, blocks the cytoplasmic opening of the channel.…”

“…The recently determined structure of the inhibitory complex of GlnK (a P II protein) with the ammonia channel AmtB of Escherichia coli (14,15) showed that the extended T-loop blocks the cytoplasmic opening of the ammonia channel, explaining channel function inhibition. This structure sheds no light on the P II -NAGK complex because P II activates NAGK (6,7) and because ADP was found in the GlnK-AmtB complex (15), whereas ADP prevents P II -NAGK complex formation (6).…”

Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB

“…Two partially stacked phenyl rings, F107 and F215 in AmtB, are seen at the periplasmic pore entrance in all Amt structures (4,5,24,25) and appear to severely constrict the substrate pathway (Fig. 3).…”

Modeling the role of covalent enzyme modification inEscherichia colinitrogen metabolism

“…A more recent experiment [36] found that ammonium was not toxic at realistic physiological concentrations. However, ammonium cannot diffuse through the cell membrane and E. coli cells are known to deactivate the membrane ammonium transporter AmtB when exposed to a high-ammonium environment [37–39]. This may explain why the experiment in [36] did not see a detrimental effect of ammonium on initial growth rates, while other experiments have seen an effect of ammonium on the viability of E. coli after long exposures [28, 29].…”