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Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues.

ABSTRACT: PII proteins comprise an ancient superfamily of signal transduction proteins, widely distributed among all domains of life. In general, PII proteins measure and integrate the current carbon/nitrogen/energy status of the cell through interdependent binding of ATP, ADP and 2-oxogluterate. In response to effector molecule binding, PII proteins interact with various PII-receptors to tune central carbon- and nitrogen metabolism. In cyanobacteria, PII regulates, among others, the key enzyme for nitrogen-storage, N-acetyl-glutamate kinase (NAGK), and the co-activator of the global nitrogen-trascription factor NtcA, the PII-interacting protein-X (PipX). One of the remarkable PII variants from Synechococcus elongatus PCC 7942 that yielded mechanistic insights in PII-NAGK interaction, is the NAGK-superactivating variant I86N. Here we studied its interaction with PipX. Another critical residue is Lys58, forming a salt-bridge with 2-oxoglutarate in a PII-ATP-2-oxoglutarate complex. Here, we show that Lys58 of PII protein is a key residue for mediating PII interactions. The K58N mutation not only causes the loss of 2-oxogluterate binding but also strongly impairs binding of ADP, NAGK and PipX. Remarkably, the exchange of the nearby Leu56 to Lys in the K58N variant partially compensates for the loss of K58. This study demonstrates the potential of creating custom tailored PII variants to modulate metabolism.

SUBMITTER: Selim KA

PROVIDER: S-EPMC6908673 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues.

Selim Khaled A KA Haffner Michael M Watzer Björn B Forchhammer Karl K

Scientific reports 20191212 1

PII proteins comprise an ancient superfamily of signal transduction proteins, widely distributed among all domains of life. In general, PII proteins measure and integrate the current carbon/nitrogen/energy status of the cell through interdependent binding of ATP, ADP and 2-oxogluterate. In response to effector molecule binding, PII proteins interact with various PII-receptors to tune central carbon- and nitrogen metabolism. In cyanobacteria, PII regulates, among others, the key enzyme for nitrog ...[more]

PMID: 31831819

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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

Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues.

Publications

Tuning the in vitro sensing and signaling properties of cyanobacterial PII protein by mutation of key residues.

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