Dataset Information

Mutational analysis of the cyanobacterial nitrogen regulator PipX.

ABSTRACT: PipX provides a functional link between the cyanobacterial global transcriptional regulator NtcA and the signal transduction protein P(II), a protein found in all three domains of life as integrators of signals of the nitrogen and carbon balance. PipX, which is toxic in the absence of P(II), can form alternative complexes with NtcA and P(II) and these interactions are respectively stimulated and inhibited by 2-oxoglutarate, providing a mechanism by which P(II) can modulate expression at the NtcA regulon. Structural information on PipX-NtcA complexes suggests that PipX coactivates NtcA controlled genes by stabilizing the active conformation of NtcA bound to 2-oxoglutarate and by possibly helping recruit RNA polymerase. To get insights into PipX functions, we perform here a mutational analysis of pipX informed by the structures of PipX-P(II) and PipX-NtcA complexes and evaluate the impact of point mutations on toxicity and gene expression. Two amino acid substitutions (Y32A and E4A) were of particular interest, since they increased PipX toxicity and activated NtcA dependent genes in vivo at lower 2-oxoglutarate levels than wild type PipX. While both mutations impaired complex formation with P(II), only Y32A had a negative impact on PipX-NtcA interactions.

SUBMITTER: Laichoubi KB

PROVIDER: S-EPMC3340408 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Mutational analysis of the cyanobacterial nitrogen regulator PipX.

Laichoubi Karim Boumediene KB Espinosa Javier J Castells Miguel Angel MA Contreras Asunción A

PloS one 20120430 4

PipX provides a functional link between the cyanobacterial global transcriptional regulator NtcA and the signal transduction protein P(II), a protein found in all three domains of life as integrators of signals of the nitrogen and carbon balance. PipX, which is toxic in the absence of P(II), can form alternative complexes with NtcA and P(II) and these interactions are respectively stimulated and inhibited by 2-oxoglutarate, providing a mechanism by which P(II) can modulate expression at the NtcA ...[more]

PMID: 22558239

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Project description:Cyanobacteria, phototrophic organisms that perform oxygenic photosynthesis, perceive nitrogen status by sensing 2-oxoglutarate levels. PII, a widespread signaling protein, senses and transduces nitrogen and energy status to target proteins, regulating metabolism and gene expression. In cyanobacteria, under conditions of low 2-oxoglutarate, PII forms complexes with the enzyme N-acetyl glutamate kinase, increasing arginine biosynthesis, and with PII-interacting protein X (PipX), making PipX unavailable for binding and co-activation of the nitrogen regulator NtcA. Both the PII-PipX complex structure and in vivo functional data suggested that this complex, as such, could have regulatory functions in addition to PipX sequestration. To investigate this possibility we performed yeast three-hybrid screening of genomic libraries from Synechococcus elongatus PCC7942, searching for proteins interacting simultaneously with PII and PipX. The only prey clone found in the search expressed PlmA, a member of the GntR family of transcriptional regulators proven here by gel filtration to be homodimeric. Interactions analyses further confirmed the simultaneous requirement of PII and PipX, and showed that the PlmA contacts involve PipX elements exposed in the PII-PipX complex, specifically the C-terminal helices and one residue of the tudor-like body. In contrast, PII appears not to interact directly with PlmA, possibly being needed indirectly, to induce an extended conformation of the C-terminal helices of PipX and for modulating the surface polarity at the PII-PipX boundary, two elements that appear crucial for PlmA binding. Attempts to inactive plmA confirmed that this gene is essential in S. elongatus. Western blot assays revealed that S. elongatus PlmA, irrespective of the nitrogen regime, is a relatively abundant transcriptional regulator, suggesting the existence of a large PlmA regulon. In silico studies showed that PlmA is universally and exclusively found in cyanobacteria. Based on interaction data, on the relative amounts of the proteins involved in PII-PipX-PlmA complexes, determined in western assays, and on the restrictions imposed by the symmetries of trimeric PII and dimeric PlmA molecules, a structural and regulatory model for PlmA function is discussed in the context of the cyanobacterial nitrogen interaction network.

Dataset Information

Mutational analysis of the cyanobacterial nitrogen regulator PipX.

Publications

Mutational analysis of the cyanobacterial nitrogen regulator PipX.

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