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Research Themes Membrane proteins

Blocking AmtB

PSI-SGKB [doi:10.1038/fa_psisgkb.2008.4]
Featured Article - September 2008
Short description: Proc Natl Acad Sci USA 104, 42-47 (2007)

The PII signal transduction proteins are key players in metabolic regulation and are widely conserved amongst bacteria as well as some archaea and organelles in eukaryotic phototrophs. GlnK, one of the three groups of PII proteins, has many attributes of other PII proteins, including a conserved structure and the fact that it forms homotrimers. In several bacterial genomes, glnK genes are found linked to amtB, the ammonia transporter, and are involved in regulation of nitrogen metabolism at multiple levels. GlnK binds the AmtB transporter when a nitrogen source is abundant and directly inhibits ammonia transport. In addition GlnK binds and inhibits GlnA, the glutamine synthetase, under conditions of good nitrogen supply. GlnK is itself regulated, notably by the metabolite 2-KG, which overcomes its inhibition of GlnA and relieves binding to AmtB, allowing ammonia uptake and assimilation. In addition, post-translational modification regulates GlnK's ability to regulate its target. In Escherichia coli this takes the form of uridylylation of GlnK residue Y51 which prevents inhibition of AmtB, and in cyanobacteria phosphorylation of S46 may have a similar effect. In addition PII signal transducers are known to bind ATP and in the case of GlnK this augments its ability to inhibit the AmtB transporter.

Two independent studies shed light on GlnK inhibition of AmtB by solving the crystal structure of the co-complex. Stroud and colleagues (PNAS 10442–47 (2007)) solved the structure to 1.96 Å, while Winkler and colleagues (PNAS 1041213–18 (2007)) did so to 2.5 Å. In both cases GlnK was seen to bind in the trimeric form to the AmtB homotrimer. Key to the interaction is the T-loop of GlnK, the base of which forms the nucleotide interaction site with B- and C-loops at the interfaces of monomers in the GlnK trimer. In the complex the T-loop of each GlnK monomer extends into cytoplasmic vestibule of each AmtB monomer. While additional ammonia molecules are seen in AmtB compared to the non-GlnK bound structure and ordering is seen on the cytosolic face of the transporter as well as the GlnK T-loop itself, the overall structures of the AmtB and GlnK are similar in the complex to their unbound forms. The T-loop insert brings R47 into the transporter vestibule, blocking the AmtB channel and suggesting that this residue directly inhibits transport. The papers also suggest why uridylylation at Y51 inhibits AmtB function: this modification would sterically prevent this residue's predicted intimate interaction with the transporter. While neither study supplied ADP, surprisingly it was this rather than ATP that was found at the interfaces of the GlnK monomers, and Stroud and colleagues suggest that either a trace contaminant with ATPase activity was present, or that the GlnK complex itself may be hydrolyzing the ATP, a possibility hinted at by the identity of residues surrounding the ADP but that awaits further study. Stroud and colleagues also gained potential insights into regulation by the 2-KG metabolite, using docking to suggest that its binding might affect the conformation of the T-loop and thus affect interaction with AmtB. Altogether the structures provide a number of insights into the mechanism underlying ammonia transport inhibition by GlnK, as well the complex regulation of their interaction.

Sabbi Lall

References

  1. Franz Gruswitz, Joseph O'Connell, III and Robert M. Stroud Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96 Å.
    Proc Natl Acad Sci USA 104, 42-47 (2007). doi:10.1073/pnas.0609796104

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