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Abstract
PII proteins are pivotal players in the control of nitrogen metabolism in bacteria and archaea, and are also found in the plastids of plants. PII proteins control the activities of a diverse range of enzymes, transcription factors and membrane transport proteins, and their regulatory effect is achieved by direct interaction with their target. Many, but by no means all, PII proteins are subject to post-translational modification of a residue within the T-loop of the protein. The protein’s modification state is influenced by the cellular nitrogen status and in the past this has been considered to regulate PII activity by controlling interaction with target proteins. However, the fundamental ability of PII proteins to respond to the cellular nitrogen status has been shown to be dependent on binding of key effector molecules, ATP, ADP, and 2-oxoglutarate which brings into question the precise role of post-translational modification. In this study we have used the Escherichia coli PII protein GlnK to examine the influence of post-translational modification (uridylylation) on the interaction between GlnK and its cognate target the ammonia channel protein AmtB. We have compared the interaction with AmtB of wild-type GlnK and a variant protein, GlnKTyr51Ala, that cannot be uridylylated. This analysis was carried out both in vivo and in vitro and showed that association and dissociation of the GlnK–AmtB complex is not dependent on the uridylylation state of GlnK. However, our in vivo studies show that post-translational modification of GlnK does influence the dynamics of its interaction with AmtB.
Highlights
Proteins of the PII signal transduction superfamily play a major role in coordinating the regulation of nitrogen metabolic processes, and have recently been implicated in regulation of at least one facet of carbon metabolism (Huergo et al, 2013; Rodrigues et al, 2014)
In this study we describe experiments to examine this topic and show that association/dissociation of the GlnK–AmtB complex can respond to the cellular N status in the absence of post-translational modification of GlnK
We have employed a combination of in vitro and in vivo studies to investigate the role of GlnK uridylylation with respect to the interaction of GlnK with its primary target, the ammonium transport protein AmtB
Summary
Proteins of the PII signal transduction superfamily play a major role in coordinating the regulation of nitrogen metabolic processes, and have recently been implicated in regulation of at least one facet of carbon metabolism (Huergo et al, 2013; Rodrigues et al, 2014) They mediate their effects by protein– protein interaction and their targets include key metabolic and regulatory enzymes, transcription factors, and nutrient transporters. Studies of E. coli GlnK indicate that in the absence of 2-OG the bound ATP is subject to PII-mediated hydrolysis to ADP (Radchenko et al, 2013) This in turn leads to a rearrangement of particular residues in the GlnK binding pocket, most notably Gln and Lys, and a concomitant change in the T-loop to form a more defined structure such that the apex of the loop projects very markedly above the protein’s surface (Conroy et al, 2007; Truan et al, 2010). The switch from the MgATP, 2-OG form of PII in N-limited conditions to the ADP-bound form in N-sufficient conditions is reflected in a concomitant change in T-loop structure which could potentially be sufficient to regulate the ability of PII proteins to interact with their cognate targets
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