Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity
- Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, United States
- Department of Biochemistry. University of Washington, United States
- Institut Pasteur, Université Paris Cité, CNRS UMR 3528, Computational Structural Biology Unit, France
- Department of Chemistry and Biochemistry, San Francisco State University, United States
- Department of Biological Sciences, KAIST, Republic of Korea
Figures
Figure 1 with 4 supplements
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 cryo-electron microscopy (cryo-EM) 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. Kinetic constants were determined from n=3 technical replicates. (D) Cryo-EM reconstruction of apo-GS filament form. Decamers are rotated relative to each other by ~26 degrees. The minimal fivefold filament interface formed consists of K52, C53, and E55 of adjacent monomers from different decamers. No obvious connecting cryo-EM density is observed in the apo map. Cross Subunit Distances that are less than 10 Å between cysteine residues or lysine residues are listed as potential crosslinking sites for bifunctional crosslinkers Bis-sulfosuccinimidyl glutarate (BSG) or bis-maleimoethane (BMOE) (E) Bifunctional crosslinking with BSG (top) or BMOE (bottom) to covalently attached adjacent primary amines (K52) or cysteines (C53), respectively followed by SDS-PAGE reveals crosslinked species only in the wild-type GS protein sample, but not K52A or C53A indicating presence and disruption of filament formation, respectively.
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Figure 1—source data 1
Labeled full uncropped gels for the crosslinking experiments shown in Figure 1E.
Zipped folder containing annotated images of complete gels: the three bis-maleimoethane (BMOE) crosslinking gels (stain-free detection; wild-type, K52A, and C53A) that appear in Figure 1E. Molecular weight standards and the crosslinked glutamine synthetase (GS) dimer and GS monomer bands are indicated on every gel. No part of any gel image has been cropped, retouched, or otherwise altered.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for the full raw uncropped, unedited gels displayed in Figure 1E.
Zipped folder containing the unedited scans of gels as captured, with no cropping, contrast adjustment or annotation: the three bis-maleimoethane (BMOE) crosslinking gels of Figure 1E as full-resolution imager exports.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig1-data2-v1.zip
Figure 1—figure supplement 1
Biochemical characterization of recombinant human glutamine synthetase (GS).
(A) 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 KMglutamate is noted to be threefold 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. Kinetic constants were determined from n=3 technical replicates. (B) 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 Superose 6 increase (Cytiva) column which corresponds to the decameric species. Molecular weight calibration curve and predicted molecular weights are included below. (C) Steady-state NADH coupled-enzyme assays were performed to validate enzymatic coupling in the presence and absence of TCEP. In all cases, robust ADP production for GS was only observed when all three substrates were present. Kinetic traces were determined from n=3 technical replicates. (D) Full SDS-PAGE gel images for bifunctional crosslinking experiments presented in Figure 1E using Bis-sulfosuccinimidyl glutarate (BSG; left) or bis-maleimoethane (BMOE; right).
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Figure 1—figure supplement 1—source data 1
Steady-state kinetic data comparing His6-tagged and scarless glutamine synthetase (GS), and NADH-coupled assay controls.
Source data for Figure 1—figure supplement 1A and C.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Labeled full uncropped gels for the crosslinking experiments shown in Figure 1—figure supplement 1D.
Zipped folder containing annotated images of complete gels: the bis-sulfosuccinimidyl glutarate (BSG) crosslinking gel (Coomassie detection) and the bis-maleimoethane (BMOE) crosslinking gel (stain-free detection) that appear in Figure 1—figure supplement 1D. Molecular weight standards and the crosslinked glutamine synthetase (GS) dimer and GS monomer bands are indicated on every gel. No part of any gel image has been cropped, retouched, or otherwise altered.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig1-figsupp1-data2-v1.zip
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Figure 1—figure supplement 1—source data 3
Original files for the full raw uncropped, unedited gels displayed Figure 1—figure supplement 1D.
Zipped folder containing the unedited scans of gels as captured, with no cropping.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig1-figsupp1-data3-v1.zip
Figure 1—figure supplement 2
Cryo-electron microscopy (cryo-EM) 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.
Figure 1—figure supplement 3
Sharpened cryo-electron microscopy (cryo-EM) density (see Methods) for the apo-filament is presented for four regions of the map containing either β-sheet (residues 40–47 and 130–140) or α-helix (residues 174–187 and 211–233) to demonstrate side chain density and fit.
Figure 1—figure supplement 4
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).
Figure 2 with 9 supplements
Glutamine binds to glutamine synthetase (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 cryo-electron microscopy (cryo-EM) 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 cryo-EM 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) Cryo-EM screening data demonstrating robust filament seeding from addition of ATP and glutamine to human GS prior to vitrification. (D) Turnover-filament global 3D reconstruction (left) and locally refined filament interface (right) 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 technical replicates) see Methods.
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Figure 2—source data 1
Decamer and filament particle counts from the time-resolved cryo-electron microscopy (cryo-EM) datasets.
Particle numbers and derived filament fractions at each quench time, used for the quantification in Figure 2B.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig2-data1-v1.xlsx
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Figure 2—source data 2
Steady-state coupled-ATPase progress curves for wild-type glutamine synthetase (GS) with and without glutamine.
Absorbance traces converted to ADP produced, used for the global fits reported in Figure 2E.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig2-data2-v1.xlsx
Figure 2—figure supplement 1
Cryo-electron microscopy (cryo-EM) data processing pipeline for initial low resolution turnover-filament.
Cryo-EM 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.
Figure 2—figure supplement 2
Cryo-electron microscopy (cryo-EM) data processing pipeline for time-resolved datasets and turnover-decamer map.
(A) 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 57 s turnover dataset was for 3D reconstruction of the decamer turnover map and displays the utilized 2D classes, 3D reconstruction details for the final map (unsharpened) with associated particle counts. (B) FSC curves for the final turnover decamer map. (C) Local resolution estimation turnover decamer map. (D) Active site ADP and Mg(II) density (sharpened) depiction at threshold 12 for chain A (left) and chain G (right).
Figure 2—figure supplement 3
Global 2D classification of time-resolved datasets.
Representative template-picked particle images for the 57 s and 517 s datasets are depicted above. All five tr-cryo-electron microscopy (cryo-EM) 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 particle classes were then referenced to the original dataset using cryoSPARC tools and the fraction of each species was quantified and linearly fit (bottom right).
Figure 2—figure supplement 4
Global 2D classification of time-resolved datasets with more stringent particle picks for quantification.
All five tr-cryo-electron microscopy (cryo-EM) 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. 2D classes representing the ‘top’ view (showing all five pentamer edges) were not quantified as these could not be confidently related to either a filament or decamer class. The filament and decamer particles classes were then referenced to the original dataset using cryoSPARC tools and the fraction of each species was quantified and linearly fit (right).
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Figure 2—figure supplement 4—source data 1
Decamer and filament particle counts from global 2D classification with stringent particle picking.
Source data for Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig2-figsupp4-data1-v1.xlsx
Figure 2—figure supplement 5
Cryo-electron microscopy (cryo-EM) data processing pipeline for turnover-filament map and associated ligand evaluation at filament interface.
(A) Cryo-electron microscopy (cryo-EM) 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 map (sharpened) with associated particle counts. Below are local resolution estimation map (unsharpened) and FSC curves for the final map. (B) Local refinement of the filament interface was conducted by symmetry expanding the particles used in the global refinement by their D5 symmetry operator, constructing a mask centered on one glutamine-bound filament interface and including portions of four neighboring subunits before local refinement was conducted without symmetry. (C) Glutamine synthetase (GS) products (glutamine, PO4, and ADP) individually fit into the filament interface density of the global refined map and for the locally refined map. Each map was sharpened per procedure in Methods and correlation values (CC) and Qscore values are included for each fit.
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Figure 2—figure supplement 5—source data 1
Decamer and filament particle counts for the high-resolution turnover-filament reconstruction.
Source data for Figure 2—figure supplement 5.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig2-figsupp5-data1-v1.xlsx
Figure 2—figure supplement 6
Sharpened cryo-electron microscopy (cryo-EM) density (see Methods) for the turnover filament dataset is presented for four regions of the map containing either β-sheet (residues 40–47 and 130–140) or α-helix (residues 174–187 and 211–233) to demonstrate side chain density and fit.
Figure 2—figure supplement 7
Steady-state kinetic screening with Michaelis–Menten fitting for wild-type, K52A, and C53A glutamine synthetase (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. Kinetic constants were determined from n=1 for 0.1 mg/mL enzyme concentration due to enzyme yield limitations and n=3 technical replicates for 0.01 mg/mL enzyme concentration and were treated as diagnostic regarding oligomeric state influence on kinetic constant profiles.
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Figure 2—figure supplement 7—source data 1
Steady-state Michaelis–Menten data for wild-type, K52A, and C53A GS.
Initial rates across ATP and glutamate concentrations for each variant, used for the fits in Figure 2—figure supplement 7.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig2-figsupp7-data1-v1.xlsx
Figure 2—figure supplement 8
Initial assessment of filament steady-state kinetics.
(A) Mass photometry assessment of oligomeric state for wild-type glutamine synthetase (GS) at 0.01 mg/mL demonstrating presence of 2-decameric enzyme in the 2-decameric size-exclusion chromatography (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 twofold change in KM,ammonia. An approximate sigma value of 30 μM was used for fitting each dataset.
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Figure 2—figure supplement 8—source data 1
Coupled-ATPase progress curves for the 2-decamer and decamer size-exclusion peak fractions.
Source data for Figure 2—figure supplement 8.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig2-figsupp8-data1-v1.xlsx
Figure 2—figure supplement 9
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 glutamine synthetase (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. Raw data are included in Figure 2—source data 2.
Figure 3 with 6 supplements
Variable E305-loop density and conformations in human glutamine synthetase.
(A) Turnover-decamer cryo-electron microscopy (cryo-EM) density map is colored based on monomer except for one E-305 loop which 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) Cryo-EM 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 (Figure 3—figure supplement 3). (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 cryo-EM 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.
Figure 3—figure supplement 1
Sharpened cryo-electron microscopy (cryo-EM) density (see Methods) for the ATP-filament is presented for four regions of the map containing either β-sheet (residues 40–47 and 130–140) or α-helix (residues 174–187 and 211–233) to demonstrate side chain density and fit.
Figure 3—figure supplement 2
Sharpened cryo-electron microscopy (cryo-EM) density (see Methods) for the turnover decamer is presented for four regions of the map containing either β-sheet (residues 40–47 and 130–140) or α-helix (residues 174–187 and 211–233) to demonstrate side chain density and fit.
Figure 3—figure supplement 3
Relative loop density correlations between unsharpened cryo-electron microscopy (cryo-EM) maps.
Comparison of cryo-EM 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.
Figure 3—figure supplement 4
Turnover decamer focused classification reveals partial E-flap density.
Pooled turnover decamer particles from the full time-resolved aggregate data were refined with C5 symmetry before symmetry expansion and focused classification on the asymmetric unit. The E-flap from PDB:2QC8 residues 290–312 (red) are fit and overlaid with each density. Four classes (3, 7, 8, and 12) were identified as having partial E-flap density, starred above and focused below, whereas all other classes were largely devoid of E-flap density.
Figure 3—figure supplement 5
Turnover filament focused classification does not recover partial E-flap density.
Turnover filament particles were symmetry expanded and subjected to focused classification on the asymmetric unit. The E-flap from PDB:2QC8 residues 290–312 (red) are fit and overlaid with each density. However, no classes were identified as having partial E-flap density whereas all other classes were largely devoid of E-flap density.
Figure 3—figure supplement 6
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.
Figure 4 with 1 supplement
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 cryo-electron microscopy (cryo-EM) 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.
Figure 4—figure supplement 1
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.
Figure 5 with 3 supplements
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). Kinetic constants were determined from n=3 technical replicates. (B) Cryo-electron microscopy (cryo-EM) 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. Cell proliferation assays were performed in technical quadruplicate; a single clone of the landing pad cell line was generated, and a single population of each GLUL variant cell line was created for testing.
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Figure 5—source data 1
Steady-state kinetic data for wild-type GS and the L300A, I309A, R298A, D239A, H304A, and E305A variants.
Initial rates across ATP, glutamate and ammonia concentrations for each variant, used for the kinetic constants reported in Figure 5A.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig5-data1-v1.xlsx
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Figure 5—source data 2
Cell proliferation data for wild-type, P242stop, R298A, and L300A GS cell lines.
CellTiter-Glo luminescence under glutamine-replete and glutamine-deplete conditions, used for Figure 5C.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Cryo-electron microscopy (cryo-EM) processing pipeline for R298A turnover-decamer and comparison to wild-type turnover decamer.
(A) Depicted in the pipeline includes grid type, protein concentration, representative micrographs, utilized 2D classes, 3D reconstruction details, and final map (unsharpened) with associated particle counts. (B) Local resolution estimation map (unsharpened). (C) FSC curves for the final map. (D) Active site ADP and Mg(II) density (sharpened) depiction at threshold 2.5 for chain A (left) and chain G (right).
Figure 5—figure supplement 2
Sharpened cryo-electron microscopy (cryo-EM) density (see Methods) for the R298A turnover decamer is presented for four regions of the map containing either β-sheet (residues 40–47 and 130–140) or α-helix (residues 174–187 and 211–233) to demonstrate side chain density and fit.
Figure 5—figure supplement 3
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 glutamine synthetase (GS)-dependent growth rates for wild-type, C53A, and K52A variants, while P242stop GS could not support cell growth. Cell proliferation assays were performed in technical quadruplicate; a single clone of the landing pad cell line was generated, and a single population of each GLUL variant cell line was created for testing.
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Figure 5—figure supplement 3—source data 1
Cell proliferation data for wild-type, P242stop, K52A, and C53A GS cell lines.
CellTiter-Glo luminescence under glutamine-replete and glutamine-deplete conditions, used for Figure 5—figure supplement 3.
- https://cdn.elifesciences.org/articles/108336/elife-108336-fig5-figsupp3-data1-v1.xlsx
Figure 6
Filament formation tunes E305-loop dynamics to modulate enzyme activity.
Left: cryo-electron microscopy (cryo-EM) 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 glutamine synthetase (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.
Scheme 1
Steady-state Michaelis-Menten kinetic model.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Homo sapiens) | GLUL | UniProt | P15104 | |
| Strain, strain background (Escherichia coli) | BL21(DE3) | New England Bioscience | C2527H | Chemical comp cells |
| Cell line (H. sapiens) | HEK293E GLUL-/- | PMID:29359789 | ||
| Cell line (H. sapiens) | HEK293E KO-TetBxb1BFP | This study | Clone 15 | Verified by glutamine auxotrophy that was recovered by DOX induction of wild-type GLUL after integration into landing pad. Available on request from the Fraser lab, UCSF (see Materials availability). |
| Cell line (H. sapiens) | HEK293E GLUL-variants (wild-type, C53A, K52A, R298A, L300A, P242stop) | This study | Clone 15 | Stable GLUL-variant lines recombined into the GS-KO-TetBxb1BFP landing pad clone; identity confirmed by mCherry expression. Available on request from the Fraser lab, UCSF (see Materials availability). |
| Recombinant DNA reagent | pDEST hsGS wild-type and associated variants | This paper | His6-SUMO tagged GLUL variants for E. coli expression. Available on request from the Fraser lab, UCSF (see Materials availability). | |
| Recombinant DNA reagent | Intein-CBD-His6-GS | This paper | GS bearing a C-terminal Mxe GyrA intein and chitin-binding domain with a 3' His6 tag, for scarless GS production in E. coli by DTT-induced self-cleavage. Available on request from the Fraser lab, UCSF (see Materials availability). | |
| Recombinant DNA reagent | pFGET1 – Ulp1 | Addgene | RRID:Addgene_64697 | His6-Ulp1 protease expression plasmid |
| Recombinant DNA reagent | pBxb1 | PMID:31612958 | Bxb1 recombinase expression plasmid | |
| Recombinant DNA reagent | attB-GLUL-IRES-mCherry | This study | Integration and expression plasmid for GLUL. Wildtype and associated variants. Available on request from the Fraser lab, UCSF (see Materials availability). | |
| Chemical compound, drug | BSG (Bis-sulfosuccinimidyl glutarate) | ThermoFisher | 22323 | Amine-to-amine crosslinker |
| Chemical compound, drug | BMOE (bis-maleimidoethane) | ThermoFisher | 21610 | Sulfhydryl-to-sulfhydryl crosslinker |
| Software, algorithm | CryoSPARC | PMID:28165473 | RRID:SCR_016501 | |
| Software, algorithm | ChimeraX | PMID:37774136 | RRID:SCR_015872 | |
| Software, algorithm | Phenix | PMID:31588918 | RRID:SCR_014224 | |
| Software, algorithm | ISOLDE | PMID:29872003 | RRID:SCR_025577 | |
| Software, algorithm | Coot | PMID:31730249 | RRID:SCR_014222 | |
| Software, algorithm | GROMACS | PMID:33032406 | RRID:SCR_014565 | |
| Software, algorithm | PLUMED | PMID:31363226 | RRID:SCR_021952 |
Additional files
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Supplementary file 1
cryo-electron microscopy (cryo-EM) data collection, refinement, and validation statistics.
Data collection parameters, reconstruction statistics, and model refinement and validation metrics for the apo filament (PDB 9OTQ, EMD-70845), turnover filament (PDB 9OTM, EMD-70841), turnover decamer (PDB 9OTO, EMD-70843), ATP filament (PDB 9OTN, EMD-70842), and R298A turnover decamer (PDB 9OTP, EMD-70844).
- https://cdn.elifesciences.org/articles/108336/elife-108336-supp1-v1.docx
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Supplementary file 2
Ligand model statistics for the deposited structures.
Cross-correlation (CC), Q-score, and B-factor for every modelled ligand in the five deposited structures. CC and Q-score were measured with models fit into sharpened maps in ChimeraX; B-factors were derived from Phenix and deposited with the structures.
- https://cdn.elifesciences.org/articles/108336/elife-108336-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108336/elife-108336-mdarchecklist1-v1.docx
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Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity
eLife 14:RP108336.
https://doi.org/10.7554/eLife.108336.3
