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High complexity of Glutamine synthetase regulation in Methanosarcina mazei : Small protein 26 interacts and enhances glutamine synthetase activity
Author(s) -
Gutt Miriam,
Jordan Britta,
Weidenbach Katrin,
Gudzuhn Mirja,
Kiessling Claudia,
Cassidy Liam,
Helbig Andreas,
Tholey Andreas,
Pyper Dennis Joshua,
Kubatova Nina,
Schwalbe Harald,
Schmitz Ruth Anne
Publication year - 2021
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.15799
Subject(s) - methanosarcina , glutamine synthetase , biochemistry , microscale thermophoresis , amino acid , biology , glutamine , glutamine amidotransferase , archaea , gene
Small ORF (sORF)‐encoded small proteins have been overlooked for a long time due to challenges in prediction and distinguishing between coding‐ and noncoding‐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 on nucleotide level as well as on the coded amino acids within numerous Methanosarcina strains strongly arguing for a cellular function of the small protein. spRNA26 level is significantly enhanced under nitrogen limitation, but also under oxygen and salt stress conditions. Using heterologously expressed and purified sP26 in independent biochemical approaches [pull‐down by affinity chromatography followed by MS analysis, reverse pull‐down, microscale thermophoresis, size‐exclusion chromatography, and nuclear magnetic resonance spectroscopy (NMR) analysis], we observed that sP26 interacts and forms complexes with M. mazei glutamine synthetase (GlnA 1 ) with high affinity (app. K D = 0.76 µ m ± 0.29 µ m ). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA 1 . Upon interaction with sP26, GlnA 1 activity is significantly stimulated, independently and in addition to the known activation by the metabolite 2‐oxoglutarate (2‐OG). Besides, strong interaction of sP26 with the PII‐like protein GlnK 1 was demonstrated (app. K D = 2.9 µ m ± 0.9 µ m ). On the basis of these findings, we propose that in addition to 2‐OG, sP26 enhances GlnA 1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA 1 .