Figures and data
GlnA1-dodecamer-assembly is induced by 2-OG without detectable oligomeric intermediates.
Oligomerisation states of purified strep-tagged GlnA1 were assessed in dependence of 2-OG by mass photometry as described in MM using a Refeyn twoMP mass photometer (Refeyn Ltd., Oxford, UK). Mass spectra are shown with relative counts (number of counts in relation to the total counts) plotted against the molecular weight. A: 75 nM GlnA1 were preincubated in the presence of varying 2-OG concentrations (0 to 25 mM) for ten min at room temperature and kept on ice until measurement. The percentage of dodecamer considering the total number of counts was plotted against the 2-OG concentration. One out of two independent biological replicates with each three technical replicates is shown exemplarily. The molecular masses shown above the peaks correspond to a Gaussian fit of the respective peak (Gaussian fit not shown) and the KD is indicated in green. B: Exemplary mass spectra of GlnA1 oligomers in the presence of 0.1 and 12.5 mM 2-OG. C: Mass spectra of the three technical replicates (different green colors) of GlnA1-oligomers at 0.39, 0.78 and 1.56 mM 2-OG, excluding the presence of intermediates. D: The specific activity of purified strep-tagged GlnA1 was determined as described in MM in the presence of varying 2-OG concentrations (0, 1.25, 5 and 12.5 mM). The standard deviation of four technical replicates is indicated for one out of two biological replicates.
GlnA1-dodecamer-assembly and activity are not influenced by GlnK1 under the conditions tested.
Purified strep-tagged GlnA1 and tag-less GlnK1 were incubated in the absence or presence of 2-OG in varying concentrations for ten min at RT. Oligomerisation states were assessed by mass photometry. Mass spectra are shown with relative counts (see Fig. 1). A: The obtained ratio of GlnA1 dodecamer/dimer of three technical replicates are shown for varying ratios between GlnA1 and GlnK1 (20:1, 2:1, 2:10, ratios relating to monomers) in the absence of 2-OG. B, C: Exemplary mass spectra of GlnA1 incubated in the absence and presence of GlnK1 (2:1) at 2-OG concentrations of 0 mM (B) and 12.5 mM (C). The molecular masses shown above the peaks correspond to a Gaussian fit of the respective peak (Gaussian fit not shown). D: 200 nM monomeric GlnA1 were preincubated with GlnK1 (in a 2:1 ratio) in the presence of varying 2-OG concentrations (0.19 to 12.5 mM) for ten min at RT. The percentage of GlnA1 dodecamer considering the total number of counts was plotted against the 2-OG concentration. One biological replicate with three technical replicates was performed. The ratio of GlnA1 dodecamer/dimer was plotted against the 2-OG concentration and the KD is indicated in green (●, -GlnK1) and yellow (●, + GlnK1). E: The specific activity of purified strep-tagged GlnA1 in the absence and presence of GlnK1 (ratio 2:1) was determined as described in MM in the presence of varying 2-OG concentrations (0, 0.78, 6.25 and 12.5 mM). The standard deviations of four technical replicates of one biological replicate are indicated.
Structure of M. Mazei GlnA1 with 2-OG.
A: Three-dimensional segmented cryo-EM density of the dodecameric complex colored by subunits. B: Corresponding views of the GlnA1 atomic model in cartoon representation.
Dimeric Interface and 2-OG binding site of dodecameric GlnA1.
A: Surface representation of the M. mazei GlnA1 2-OG dodecamer with three GlnA1 protomers fitted in cartoon representation into the dodecamer as vertical (blue and ochre) and horizontal (blue and green) dimers. B: Horizontal dimers and close-up of 2-OG binding site. Important residues are shown as atomic stick representation, primed labels indicate neighboring protomer. 2-OG and water molecules important for ligand binding fitted into density are shown in grey. Dotted lines represent polar interactions between 2-OG, waters and residues. C Vertical dimers and close-up of dimerization site. C-terminal helices H14/15 and H14’/ H15’ of two neighboring protomers lead to tight interaction, mediated by hydrophobic and polar interactions. D: Top-view of GlnA1 hexamer, 2-OG and substrate binding sites are depicted for one horizontal dimer.
Comparison of 2-OG and substrate binding site of 2-OG bound, apo and TS structures (Schumacher et al., 2023).
Atomic models in cartoon, important residues shown in stick representation. Colors: blue/green, purple/ochre and red/yellow represent M. mazei GlnA1 2-OG, M. mazei GlnA1 apo (PDB: 8tfb, Schumacher et al., 2023) and M. mazei GlnA1 Met-Sox-P·ADP (PDB: 8tfk, Schumacher et al., 2023) transition state (GlnA1 TS), respectively. A left: GlnA1 2-OG dimer in superposition with GlnA1 apo showing large scale movements upon 2-OG binding. A right: Close-up of 2-OG binding site of GlnA1 2-OG in superposition with GlnA1 apo. Dramatic movement of Helix α3 (residue 167-181) and R87 loop show effect of 2-OG binding. B: Close-up of substrate binding site of GlnA1 2-OG in superposition with GlnA1 apo and ADP ligand from GlnA1 TS. Helix α3 movement upon 2-OG binding leads to a cascade of conformational changes of the phenylalanines F184, F202 and F204 that lead to a priming of the active site for ATP binding. C: Close-up of substrate binding site of GlnA1 2-OG in superposition with GlnA1 TS shows high similarity between 2-OG bound and transition state structure. D: Close-up of substrate binding site of GlnA1 2-OG in superposition with GlnA1 apo and Met-Sox-P ligand from GlnA1 TS. Large structural changes of the D50-loop with ejection of the R66 key-residue shown. Flipping of the loop allows R319 and D57 to move in further and catalyze phosphoryl-transfer and attack of NH4+, respectively. E: Close-up of the substrate binding site of GlnA1 2-OG in in superposition with GlnA1 TS reveals strong similarity between 2-OG bound and transition state structure in the active site.
Feedback inhibition of GlnA1 by glutamine.
A: Specific activity of purified strep-tagged GlnA1 (wt) and the respective R66A-mutant protein was determined as described in Materials and Methods in the presence of 12.5 mM 2-OG and after additional supplementation of 5 mM glutamine. For wt and the R66A-mutant one out of two biological independent replicates are exemplarily shown, the deviation indicates the average of four technical replicates. B: Superposition GS structures without glutamine of M. mazei (blue, green) and B. subtilis (orange, pink; PDB: 4lnn, Murray et al., 2013): substrate binding-site including R’66 (R’62, respectively), which are responsible for feedback inhibition. C: Exemplary mass spectra of Strep-GlnA1 with 12.5 mM 2-OG in presence and absence of 5 mM glutamine. The molecular masses shown above the peaks correspond to a Gaussian fit of the respective peak (Gaussian fit not shown).
Model of the various molecular mechanisms of glutamine synthetase activity regulation.
Comparison of the regulation of glutamine synthetase activity in E. coli /Salmonella typhimurium, and B. subtilis, Synechocystis and M. mazei. GS are in general active in a dodecameric, unmodified complex under nitrogen limitation. Upon an ammonium upshift, GS are inactivated by feedback inhibition (BcGS, E. coli), covalent modification (adenylylation, EcGS) or binding of (small) inactivating proteins (Synechocystis, BsGS). M. mazei GS on the contrary is regulated via the assembly of the active dodecamer upon 2-OG-binding and furthermore is strongly feedback inhibited by glutamine. (Bolay et al., 2018; Klähn et al., 2018, 2015; Stadtman, 2001; Travis et al., 2022b). Created with BioRender.com
© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.
Affinity-purified Strep-GlnA1 and size-exclusion-chromatography (SEC) of Strep-GlnA1 after purification.
A: 1.5 µg (lane 1) and 3 µg (lane 2) Strep-GlnA1 on a coomassie-stained 12 % SDS-Gel. B: Elution profile of Strep-GlnA1 (black) and size standard (dashed line, molecular weights in italics). Size exclusion chromatography was performed on a Superose™ 6 Increase 10/300 GL column (Cytiva, Marlborough, USA) with a flow rate of 0.5 ml/min.
Sigmoidal fitted curves for mass photometry measurements of Strep-GlnA1 with varying concentrations of 2-OG.
The curves were fitted and KD-values calculated using RStudio (RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA URL). A, B: Two replicates for 2-OG titration, formation of dodecamer is shown in percent. C, D: 2-OG titration in the absence (C) and presence of GlnK1 (D), formation of dodecamer is shown as a ratio of dodecamer/dimer.
Cryo-EM Data processing workflow.
A: Representative motion-corrected micrograph showing different orientations of the GlnA1 particles B: Cryo-EM processing tree used for obtaining the high-resolution structure of GlnA1. The map obtained is coloured by resolution, where the global resolution was estimated using GSFSC C: Different regions of GlnA1 encased around the cryo-EM density.
Mass photometry of purified and thawed Strep-GlnA1 before and after SEC.
Mass spectra of Strep-GlnA1 samples with 0 and 12.5 mM after affinity-purification (blue 
Amino-acid sequence alignment of different model organism glutamine synthetases.
(Alignment tool: COBALT, visualization in SnapGene) Conserved amino-acids are highlighted in green. The relevant residues in M. mazei for 2-OG- and substrate-binding, as well as the arginine responsible for the feedback inhibition by glutamine are highlighted by coloured boxes (blue 
M. mazei GlnA1 filaments.
A: Representative motion-corrected micrograph showing GlnA1 filaments B: Reference-free 2D classes showcasing filament orientations C, D: 3D reconstructed map of GlnA1 filament and its model.
Strains and plasmids.
