High complexity of glutamine synthetase regulation in Methanosarcina mazei: 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- 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 (GlnA1) with high affinity (app. KD = 0.76 µm ± 0.29 µm). Moreover, seven amino acids were identified by NMR analysis to directly interact with GlnA1. Upon interaction with sP26, GlnA1 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 GlnK1 was demonstrated (app. KD = 2.9 µm ± 0.9 µm). On the basis of these findings, we propose that in addition to 2-OG, sP26 enhances GlnA1 activity under nitrogen limitation most likely by stabilizing the dodecameric structure of GlnA1.

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Author:Miriam Gutt, Britta Jordan, Katrin Weidenbach, Mirja Gudzuhn, Claudia Kiessling, Liam Cassidy, Andreas J. Helbig, Andreas Tholey, Dennis Joshua Pyper, Nina Kubatova, Harald SchwalbeORCiDGND, Ruth Schmitz-Streit
Parent Title (English):The FEBS journal
Place of publication:Oxford
Document Type:Article
Date of Publication (online):2021/03/04
Date of first Publication:2021/03/04
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2021/07/26
Tag:small proteins
Methanosarcina mazei; glutamine synthetase regulation; nitrogen regulation; small ORFs
Issue:Online Version of Record before inclusion in an issue
Page Number:24
First Page:1
Last Page:24
Institutes:Wissenschaftliche Zentren und koordinierte Programme / Zentrum für Biomolekulare Magnetische Resonanz (BMRZ)
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung 4.0