Human Glutamine Synthetase Forms Filaments.

A) At top, reaction mechanism of glutamine synthetase. Middle: surface representation of PDB:2QC8 ADP + Methionine sulfoximine (MSO) bound x-ray crystal structure of human glutamine synthetase. Bottom: N-terminal His6-tag perturbs structure of human glutamine synthetase (GS) as observed by 2D classification of negative stain and cryoEM data. B) Size-exclusion chromatography reveals His6-tag dramatically alters the oligomeric state of human GS and scarless GS can form higher order oligomers. C) His6-tagged GS displays strongly altered steady-state kinetic constants when compared to scarless GS. N.D. = not defined as His6-tagged GS also display inefficient coupling of ATP hydrolysis with glutamine formation and thus KM,ammonia could not be defined for this enzyme. D) cryoEM reconstruction of apo-GS filament form.

Glutamine binds to GS filaments at the interface.

A) Establishment of steady state conditions prior to vitrification with GS at low concentration (0.15 mg/mL) revealed presence of 2-decameric species (red arrows) in addition to decameric species (blue arrows) during cryoEM grid screening. Initial reconstruction of the turnover 2-decameric map demonstrated clear density connecting decamers (red dotted circle) not previously observed in the apo-filament map and loss of E305-loop density (black dotted circle). B) Time-resolved cryoEM demonstrates linear relationship between glutamine and filament formation assessed by independently processing five datasets and quantifying the fraction of decamer and filament present (see Methods) and plotted against the vitrification quench time of the reaction. C) cryoEM screening data demonstrating robust filament seeding from addition of ATP and glutamine to human GS prior to vitrification. D) Turnover-filament 3D reconstruction demonstrates similar overall architecture to apo-filament map (left) but with clear density between decamers with glutamine modeled which forms hydrogen bonds with K52, C53, and E55 (right). E) Steady-state ammonia kinetic constants for wild-type, K52A, and C53A with and without 10 mM glutamine added to reaction to stimulate filament formation. Errors were calculated as S.E.M. from global fitting and were propagated through each Michaelis-Menten parameter (Equations 2-4; n = 8 replicates) see Methods.

Variable E305-loop density and conformations in human glutamine synthetase.

A) Turnover-decamer cryoEM density map is colored based on monomer except for one E-305 loop with is colored cyan and ADP density that is colored purple demonstrating how the E305-loop closes over the active site to enclose the glutamate and ammonia binding site. B) CryoEM E305-loop density representations for the apo-filament, ATP-filament, turnover-filament, and turnover-decamer maps with 2QC8 docked into each map shown in red. The contour is chosen to equalize the relative density levels in other regions of the protein (Supplementary Figure 11). C) Representation of EMMIVox ensembles of the E305-loop (residues 295-309) for one pentamer (decamer-orange and filament-blue) overlaid with ribbon structure of consensus model (tan). (D) Single filament and decamer turnover monomers overlaid and presented in blue and orange respectively presenting a hydrophobic stabilizing patch (I285, L300, and I309) and stabilizing salt-bridge (D239 and R298) in the ‘closed active’ loop state that is missing in the filament models. Distance measurements calculated across full EMMIVox ensembles represented as kernel density function between residues participating in ‘closed active’ loop state are presented demonstrating bias away from these contacts in the turnover filament form. (E) Same as (D) but representing the ‘tip’ of the loop showing E305 hydrogen bonding with S66 and H304 hydrogen bonding with N246 with quantification of these distances from the ensemble. In all cases, the filament oligomeric state under turnover conditions disfavored these interactions. (F) MD-refinement of the cryoEM filament turnover map revealed active site loop infiltration into the active site with R299 hydrogen bonding with active site residues E136 (top left) with distance quantification of R299 to E136 in the active site (top right). At bottom, overlay of all ten monomers per MD-refined model with loops in ribbon, ADP in stick, and the rest of the model is represented in surface. The filament loop is in an outward conformation from the active site at the base of helix 8.

Global conformational change is associated with loop conformational heterogeneity under turnover conditions.

(A) Left: rotational motion about the pentameric interface observed where models were aligned on the bottom monomer and rotation seen in the upper monomer is noted (decamer in yellow and filament in blue). Middle: representation of the decameric turnover cryoEM map colored per monomer with outlines showing relevant areas shown at left and right. Right: monomeric interface within a pentamer overlay of the turnover filament model (gray) with turnover decamer model colored by monomer (tan, green, and red). Ligands are colored by atom type. Arrows indicate conformational change from filament to decamer state. The largest Ca distance change not including the E305-loop is indicated between residues 74 and 263 from across the pentamer. (B) Ca distance difference matrix for chain G between turnover decamer and turnover filament models with the E305-loop (residues 289-312) and the far C-terminal peptide (369-373) omitted with a cartoon secondary structure representation of a monomer on top and distance color legend at right demonstrating that the tip of the B-grasp region (60-76) and helix 8 (residues 262-282) showing the largest conformational differences. (C) Ca distance differences for residues 74 and 263 between the turnover filament and turnover decamer ensembles demonstrating a robust 2 angstrom distance difference.

Glutamine synthetase E305-loop conformation controls Michaelis-complex formation.

A) Steady-state kinetic parameters for wild-type GS (also displayed in Figure 2) and six point-mutants of GS, L300A, I309A, R298A, D239A, H304A, E305A as assessed by the coupled-ADP release assay (methods). (B) cryoEM density map for R298A decamer under turnover conditions displayed in full (left) with inset focused on loop density (right) demonstrating complete loss of the E305-loop for this variant. (C) Schematic of HEK293E GLUL-/- cell line auxotrophic for glutamine is reconstituted with single genomic integration of individual GLUL variants (methods) and used to assess glutamine prototrophy. All variant cell lines grow equivalently in glutamine replete media conditions (middle) but only the wild-type GLUL variant could support cell growth and survival under glutamine deplete conditions, whereas R298A, L300A, and a premature stop codon at P242 all led to cell death.

Filament formation tunes E305-loop dynamics to modulate enzyme activity.

Left: cryoEM map of the turnover filament which includes a higher KM, ammonia and open E305-loop indicated at bottom and by an ‘open’ red colored E305-loop cartoon. Also indicated is the global conformational change that opens up the monomeric interfaces about the active site (yellow lines). This form of the enzyme is increased with increased GS concentration and/or glutamine concentration and is different from the decamer which includes a more closed active site and less conformationally heterogeneous E305-loop. Right: proposed conformational landscape of GS wherein the global fold of the enzyme is largely retained and subtle conformational/ensemble changes found in the low thermodynamic basin are present. Inset: the E305-loop ensemble is depicted for the turnover decamer, turnover filament, and E305-loop mutants wherein the decamer slightly favors an active-closed state, the filament an open-ensemble, and loop variants significantly favor the open-ensemble state.

Biochemical characterization of recombinant human GS.

Left: Steady-state kinetic analysis of N-terminally His6-tagged GS (top) and tagless GS derived from the GS-Intein-CBD-His6 tagged construct where the Km(glu) is noted to be 3-fold higher for His6-tagged GS and His6-tagged GS is noted to display non-specific ATP hydrolysis under ATP + Glutamate conditions suggesting potential loss of γ-glutaryl phosphate intermediate. Right: size-exclusion chromatography traces of tagless GS constructs derived either from the His10-Sumo tagged GS or Intein-CBD-His6 tagged GS where in both cases the predominant molecular weight species is found at ∼16 mL elution volume on a superose6 increase (Cytiva) column which corresponds to the decameric species.

cryoEM data processing pipeline for apo-filament map.

Depicted in the pipeline includes grid type, protein concentration, representative micrographs, utilized 2D classes, 3D reconstruction details, and final maps with associated particle counts and FSC curves.

Negative stain EM of human glutamine synthetase variants showing attenuation of filament formation for the point mutants K52A (middle) and C53A (right) compared to wild-type (left).

cryoEM data processing pipeline for initial low resolution turnover-filament.

cryoEM data processing pipeline for low resolution turnover filament map. Depicted in the pipeline includes grid type, protein concentration, representative micrographs, utilized 2D classes, 3D reconstruction details, and final map with FSC curve.

cryoEM data processing pipeline for time-resolved datasets and turnover-decamer map.

Depicted in the pipeline includes five total datasets where in each dataset, the grid type, protein concentration, representative micrographs, and 2D processing workflow. In addition, the 57s turnover dataset was for 3D reconstruction of the decamer turnover map and displays the utilized 2D classes, 3D reconstruction details, and final maps with associated particle counts, local resolution estimation maps, and FSC curves.

Global 2D classification of time-resolved datasets.

Representative template picked particle images for the 57s and 517s datasets are depicted above. All five tr-cryoEM datasets were combined and classified in two dimensions and separated into filament, decamer, and discarded classes with 2D class images depicted at bottom left. 2D classes where partial head-on-head contacts could be discerned were included in the filament classification. 2D classes where multiple decamers were present, but not in a head-on-head arrangement were discarded. The filament and decamer particles classes were then referenced to origin dataset using cryoSPARC tools and the fraction of each species was quantified and linearly fit (bottom right).

A. CryoEM data processing pipeline for high resolution turnover-filament. Depicted in the pipeline includes grid type, protein concentration, representative micrographs, utilized 2D classes, 3D reconstruction details, and final maps with associated particle counts. Below are local resolution estimation maps and FSC curves for the final map. B. GS products (glutamine, PO4, and ADP) individually fit into filament interface density.

Steady-state kinetic screening with Michaelis-Menten fitting for wild-type, K52A, and C53A GS variants.

Initial velocities from the coupled-ATPase assay measurements are presented with fits to a basic Michaelis-Menten model included. Error was estimated from goodness of fit as S.E.M. Enzyme was assayed at 0.01 mg/mL (left) or 0.1 mg/mL (right) and sample from either the B8 (decamer) or B5 (2-decamer) fraction from size-exclusion chromatography from a Sup6 Increase column. The K52A and C53A variants displayed nearly identical Michaelis-Menten parameters as wild-type under both conditions. K52A B5 fraction was not of high enough concentration to assay at 0.1 mg/mL and thus not determined. *** indicates that the rate of conversion at 0.1 mg/mL enzyme was too fast to capture by initial velocity analysis.

Initial assessment of filament steady-state kinetics.

A) mass photometry assessment of oligomeric state for wild-type GS at 0.01 mg/mL demonstrating presence of 2-decameric enzyme in the 2-decameric SEC peak but not in the decameric SEC peak fraction. Additionally observed is the multiple oligomeric states GS could be found in solution at lower concentration. B) Steady-state product formation data for wild-type GS 2-decameric or decameric SEC peaks performed at 0.1 mg/mL GS with varied ammonia concentrations. Top, ADP product concentration (n=1 per concentration) per time at multiple ammonia concentrations with saturating glutamate and ATP globally fit to a simple Michaelis-Menten Model shown in middle using Kintek Explorer. Fit parameters displayed at bottom demonstrate a 2-fold change in KM,ammonia. An approximate sigma value of 30 μM was used for fitting each dataset.

Glutamine-stabilized filament steady-state global fitting to determine KM,ammonia.

The turnover filament map is presented in purple (chain A), black mesh (ligand of chain A), and orange (chain E). Active site residues are shown from the consensus EMMIVox model. Absence of density corresponding to the amino acid binding pocket is shown (black circle) demonstrating that despite high glutamine levels in this dataset, no glutamine is bound to the active site. Right, steady-state data is presented as an average of eight independent progress curve datasets with GS at 0.1 mg/mL, glutamate at 50 mM starting concentration, ATP at 5 mM starting concentration, and ammonium chloride at 0.75, 0.375, and 0.188 mM as indicated. Data were globally fit to a simple Michael-Menten model (bottom left) using Kintek Explorer. Fits are included in plotted data. An average sigma value of 50 μM was used for fitting each dataset.

Relative loop density correlations between cryoEM maps.

Comparison of cryoEM density maps between apo-filament, ATP filament, Turnover Decamer and Turnover Filament for Chains A and G across the pentamer:pentamer interface. Density was manually thresholded in ChimeraX to be approximately similar by side change density for residues in helix 8 (265-285). A subsegment of PDB 2QC8 (residues 265-316 which includes the E305-loop (residues 289-309)) was fit into the density using the ‘Fit in Map’ tool in ChimeraX and the residues outside the contour level are reported. In general, the apo-filament map had the most density corresponding to the E305-loop, followed by the ATP filament map, Turnover Decamer map, and Turnover Filament map.

Ensemble analysis including apo-filament dataset.

Top: RMSF violin plots depicting root mean square fluctuation (RMSF) of E305-loop (residues 299-311), Filament Interface loop (residues 48-57), or ‘top’ of bifunnel active site (residues within 4.5 Å of bound nucleotide) showing highest fluctuation for the E305-loop and within the E305-loop region, the highest RMSF is noted for the turnover filament followed by turnover decamer and these least RMSF for the apo-filament dataset. Both the filament interface loop and active site displayed low RMSF overall. Bottom: Similar to Figure 3 but including overlay of apo-filament ensemble data. MD-ensemble refinement distance quantification of S66-E305, N246-H304, D239-R298, I285-L300, and I285-L309. In all cases, the apo ensemble demonstrates a more stable E305-loop where all stabilizing interactions are maintained with higher probability.

Global conformational changes most apparent in Turnover Filament structure.

C-α distance difference matrix for chains A and G between Turnover Decamer and Turnover Filament models (top left), Apo Filament and Turnover-Filament models (top right), and Decamer Turnover and Apo Filament models (bottom left) depicted with and without the E305-loop (residues 289-312) and the far C-terminal peptide (369-373) omitted. Absolute distance color legend at right of each plot is in units of Å. These comparisons demonstrate that the tip of the B-grasp region (60-76) and helix 8 (residues 262-282) display conformational differences for the Filament Turnover model primarily. Bottom right: overlay of Turnover Filament (blue), Turnover Decamer (orange), and Apo Filament (gray) models highlighting helix 8 (residues 262-282) and showing a loss of helicity in part of helix 8 for Turnover Filament only.

cryoEM processing pipeline for R298A turnover-decamer and comparison to wild-type turnover decamer.

Depicted in the pipeline includes grid type, protein concentration, representative micrographs, utilized 2D classes, 3D reconstruction details, and final maps with associated particle counts. At the bottom left are local resolution estimation maps and FSC curves for the final map.

Glutamine auxotrophy determination of filament interface mutants.

Luminescence derived from Promega CellTiter Glo plotted against growth time for wild-type, C53A, K52A, and P242stop variants under glutamine replete (left) or deplete (right) media conditions demonstrating similar GS-dependent growth rates for wild-type, C53A, and K52A variants, while P242stop GS could not support cell growth.