Small protein 26 interacts and enhances glutamine synthetase activity inMethanosarcina mazei

Abstract

Small ORFs (sORF) encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding and non-coding predicted sORFs and in their biochemical detection and characterization. We report on the first biochemical and functional characterization of a small protein (sP26) in the archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved within numerous Methanosarcina strains on the amino acid as well as on nucleotide level strongly arguing for a cellular function of the small protein. spRNA26 is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. His-tagged sP26 was heterologously expressed and purified by fractionated ammonium sulfate precipitation, affinity chromatography and size exclusion centrifugation. Using independent biochemical approaches (pull-down by affinity chromatography followed by MS analysis, revers pull-down, microscale thermophoresis and size exclusion chromatography) we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA 1 ) with high affinity (app. KD = 45 +/-14 µM). Upon interaction with sP26, GlnA 1 activity was significantly stimulated independently and in addition to the known activation by the metabolite 2-oxoglutarate. Besides strong interaction of sP26 with the PII-like protein GlnK 1 was demonstrated (KD= 1.4 µM +/-0.9 µM). On the basis of these findings, we hypothesize that in addition to 2-oxoglutarate, sP26activates GlnA 1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA 1 .

Differences in the regulation mechanisms of the glutamine synthetase from methanogenic archaea unveiled by structural investigations

“…S2), could not be detected despite the addition of Mg 2+ or Mn 2+ , an increase of the protein concentration (up to 0.75 mg/ml final concentration) or an extension of the incubation time. As 2OG acts as an activity stimulator in archaeal GS [26][27][28] , 2OG was added to the assay and revealed to be essential for MtGS activity under these experimental conditions (Fig. 1C).…”

“…In the same archaeon, the molecule 2OG was shown to be a more efficient regulator with a 12-fold activity stimulation, reaching 18-fold when PII-family regulatory proteins GlnK1 or GlnK2 were added in addition to 2OG 26 . 2OG and GlnK also enhance the activity of the enzyme from the methanogen Methanosarcina mazei, as well as the 23 residue-large sP26 protein 27,28 , although an inhibitory effect of GlnK (under different buffer conditions) has also been shown 28 . The regulatory proteins GlnK and sP26, whose expression is triggered under nitrogen starvation, form a complex and make a tight and specific interaction with the GS 27,29 .…”

“…2OG and GlnK also enhance the activity of the enzyme from the methanogen Methanosarcina mazei, as well as the 23 residue-large sP26 protein 27,28 , although an inhibitory effect of GlnK (under different buffer conditions) has also been shown 28 . The regulatory proteins GlnK and sP26, whose expression is triggered under nitrogen starvation, form a complex and make a tight and specific interaction with the GS 27,29 . The direct control by 2OG is particularly elegant as the metabolite is a cellular sensor for nitrogen deprivation and has been shown to act on several key nodes of nitrogen acquisition in methanogens (Fig.…”

“…The direct control by 2OG is particularly elegant as the metabolite is a cellular sensor for nitrogen deprivation and has been shown to act on several key nodes of nitrogen acquisition in methanogens (Fig. S1A) 8,[26][27][28]30…”

Small but Smart: On the Diverse Role of Small Proteins in the Regulation of Cyanobacterial Metabolism

“…However, it should be noted that although IFs are unique to cyanobacteria, GS regulation by small proteins is not restricted to this group per se. For instance, GS activity is stimulated by complex formation with the 23 aa peptide sP26 and further modulated in a 2-oxoglutarate (2-OG) dependent manner by the 114 aa protein GlnK 1 in the archaeal model Methanosarcina mazei [46,47]. Both proteins are not related to the cyanobacterial IFs, which greatly exemplifies how widespread and versatile regulatory mechanisms by small proteins are even when targeting the same enzyme.…”