High complexity of Glutamine synthetase regulation in <i>Methanosarcina mazei</i> : Small protein 26 interacts and enhances glutamine synthetase activity

Small ORF (sORF)-encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding- noncoding-predicted sORFs their biochemical detection characterization. We report on the first functional characterization of protein (sP26) archaeal model organism Methanosarcina mazei, comprising 23 amino acids. The corresponding encoding leaderless mRNA (spRNA26) is highly conserved nucleotide level as well coded acids within numerous strains strongly arguing cellular function protein. spRNA26 significantly enhanced under nitrogen limitation, but also oxygen salt stress conditions. Using heterologously expressed purified sP26 independent approaches [pull-down by affinity chromatography followed MS analysis, reverse pull-down, microscale thermophoresis, size-exclusion chromatography, nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that interacts forms complexes with M. mazei glutamine synthetase (GlnA1) high (app. KD = 0.76 µm± 0.29 µm). Moreover, seven were identified NMR analysis directly interact GlnA1. Upon interaction sP26, GlnA1 activity stimulated, independently addition known activation metabolite 2-oxoglutarate (2-OG). Besides, strong PII-like GlnK1 was demonstrated 2.9 µm ± 0.9 On basis these findings, propose 2-OG, enhances limitation most likely stabilizing dodecameric structure