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.

Functional and structural characterization of PII‐like protein CutA does not support involvement in heavy metal tolerance and hints at a small‐molecule carrying/signaling role

“…A semilogarithmic plot of molecular mass (logarithmic scale) versus elution volume (linear scale) was used to estimate the oligomeric nature of Se CutA in solution. Alternatively, when indicated, a Superdex TM 200 PC 3.2/30 column (2.4 mL) was used as previously reported [7,57,58].…”

From cyanobacteria to Archaeplastida: new evolutionary insights into PII signalling in the plant kingdom

“…New Phytologist (2020) 227: 722-731 Ó 2020 The Authors New Phytologist Ó 2020 New Phytologist Trust www.newphytologist.com compete for the same sites (Fokina et al, 2010;Zeth et al, 2014;Selim et al, 2019). In most, but not all PII proteins, 2-OG binds to effector-molecule binding sites once they are occupied by Mg 2+ -ATP, as the Mg 2+ ion is an important ligand for 2-OG (Fokina et al, 2010).…”

“…Through the competitive binding of ATP and ADP and the Mg 2+ -ATP dependent binding of 2-OG, PII proteins sense the cellular energy state and the cellular C/ N state, respectively (Smith et al, 2003;Fokina et al, 2010;. Binding of the various effectors, through intricate contacts in the cleft region, results in different conformational states of PII that ultimately govern signal transduction events through direct interactions with target proteins (Forchhammer Selim et al, 2019). In prokaryotic systems, PII targets may be transcription factors, key metabolic enzymes and transporters, which together regulate the cell's metabolic status Selim et al, 2019).…”

“…Binding of the various effectors, through intricate contacts in the cleft region, results in different conformational states of PII that ultimately govern signal transduction events through direct interactions with target proteins (Forchhammer Selim et al, 2019). In prokaryotic systems, PII targets may be transcription factors, key metabolic enzymes and transporters, which together regulate the cell's metabolic status Selim et al, 2019).…”

“…New Phytologist (2020) 227: 722-731 Ó 2020 The Authors New Phytologist Ó 2020 New Phytologist Trust www.newphytologist.com ornithine/Arg biosynthesis pathway (Fig. 1) (Ll acer et al, 2008;Selim et al, 2019). NAGK is the most highly conserved target of PII in Cyanobacteria and Archaeplastida (red algae and Chlorophyta) (Sugiyama et al, 2004;Chen et al, 2006;Beez et al, 2009;Chellamuthu et al, 2013Chellamuthu et al, , 2014.…”