Expression and purification of the α1-specific 8E3-GFP Fab
Table of Contents
The α1-specific mouse monoclonal antibody 8E3 was generated and screened as previously described9. The coding sequences of 8E3 Fab light and heavy chains were determined from hybridoma mRNA, and a construct to express the Fab portion of the antibody was designed by including sequences to encode an N-terminal GP64 signal peptide. Codons were optimized for expression in insect cells. To facilitate recombinant antibody detection and purification, a 3C cleavage sequence, an EGFP gene and a twin-strep II tag were added to the C terminus of the heavy chain. Synthetic genes for both chains were then cloned into the pFastBac-Dual vector under the polyhedrin promoter. The recombinant baculovirus was prepared as previously described51. Sf9 cells at a density of 3 million per ml were infected with the recombinant baculovirus, with a multiplicity of infection of 2, and further cultured for 96 h at 20 °C. The antibody-containing supernatant was collected by a 20-min centrifugation at 5,000g and then the pH was adjusted to 8 with 30 mM Tris base, incubated in the cold room overnight to enable precipitation of non-Fab protein and clarified by another 20-min centrifugation at 5,000g. The supernatant was concentrated and buffer exchanged three times with TBS (20 mM Tris, 150 mM NaCl, pH 8) using a tangential-flow concentrator equipped with a 15 kDa filter. The concentrated supernatant was then loaded onto a 15 ml streptactin column, which was washed with at least 20 column volumes (CV) of TBS and eluted with 5 mM desthiobiotin in TBS. Selected fractions were pooled, concentrated and buffer exchanged to TBS using microconcentrators with a 50 kDa cutoff. Concentrated 8E3-GFP Fab (approximately 100 μM) was aliquoted and stored at −80 °C until use.
Purification of nα1-GABAARs from mouse brains
One-month-old BL/6 mice of mixed sex (approximately 50 mice per preparation) were used for native receptor isolation. The mice were first euthanized and decapitated. The whole brain was isolated from the skull using a laboratory micro spatula and stored in ice-cold TBS. Cerebella were removed from the whole brain, frozen in liquid nitrogen and stored at −80 °C for a separate study. After being washed twice with ice-cold TBS, brain tissue was resuspended with ice-cold TBS (1 ml per brain) supplemented with 0.2 mM phenylmethyl sulfonyl fluoride (PMSF). The suspension was processed with a loose-fit Potter–Elvehjem homogenizer for 20 full up-and-down strokes and further sonicated (1 min per 50 ml) at a setting of 6, typically at a 40 W output. The suspension was centrifuged at 10,000g for 10 min, resulting in a hard pellet of mainly the nuclear fraction and a ‘runny’ soft pellet containing a substantial amount of nα1-GABAARs. The supernatant was further centrifuged at 200,000g for 45 min to pellet the membranes. About 0.1 g of hard pellet and 0.2 g of soft pellet were obtained per mouse brain, on average. These membrane pellets were resuspended with an equal volume of TBS buffer containing protease inhibitors (aprotinin/leupeptin/pepstatin A/PMSF). If not used straightaway, the 50% membrane suspension was supplemented with 10% glycerol and snap frozen in liquid nitrogen.
The following membrane solubilization and affinity chromatography were all carried out at 4 °C. First, LMNG/CHS (10:1 w/w) stock (10% w/v) was diluted to 2.5% in TBS buffer containing protease inhibitors. Then, one volume of the 50% membrane suspension was mixed with two volumes of the diluted detergent stock and incubated for 1 h on a platform rocker, which routinely resulted in the solubilization of about 60% of the α1 subunit present in the tissue, estimated on the basis of western blot (Millipore, catalogue no. 06-868, 1:1,000 dilution) (Extended Data Fig. 1). Next, BioLock solution was added at 0.1 ml per brain to quench the naturally biotinylated proteins, and the mixture was clarified by centrifugation at 200,000g for 1 h. Finally, the 8E3-GFP Fab was added to the solubilized membrane to a concentration between 60 nM and 100 nM. After 1 h incubation, 3 ml of pre-equilibrated streptactin resin was added to bind the 8E3-GFP Fab and associated nα1-GABAARs for 2 h in batch mode.
On-column nanodisc reconstitution
MSP2N2 (ref. 52) or a recently engineered MSP1E3D1 variant, CSE3 (ref. 53), was used for on-column MSP nanodisc reconstitution. The affinity resin, bound with receptor complexes, was washed in batches, first with 20 CV of ice-cold TBS, then with 20 CV of TBS containing 0.05% LMNG and 0.01% brain polar lipid (Avanti). During this wash, 40 nmol MSP2N2 and 3.2 μmol POPC:bovine brain extract (Sigma) (85:15) lipids, or 40 nmol CSE3 and 4.8 μmol lipids, were mixed to a final volume of 1 ml in TBS and incubated at room temperature for 30 min. The beads were transferred to an empty Econo-Pac gravity flow column (Bio-Rad) to drain the buffer. Then, the 1 ml pre-incubated MSP:lipids were added and incubated for 1.5 h. Next, biobeads were added to a 20× weight excess to the LMNG detergent. The mixture was incubated with a rotator in the cold room for at least 4 h. The biobead/resin mixture was washed with 20 CV of ice-cold buffer to remove unbound empty nanodiscs.
Two approaches were used to elute reconstituted nanodiscs: competitive ligand elution and protease cleavage. For ligand elution, 0.5 CV 5 mM desthiobiotin dissolved in TBS was incubated with the streptactin superflow resin for 10 min before gravity elution, which was repeated a total of six times. In the case of 3C cleavage, 0.1 mg 3C protease was first diluted to 50 μg ml−1 with 2 ml TBS and added to the resin. After a 2 h incubation in the cold room, the elution was collected and the column was further washed three times with 2 ml TBS to improve the protein yield. 3C protease cleavage offered better protein purity and was used for the ZOL and the APG samples. Pooled elution was concentrated to about 0.5 ml using a 50 kDa cutoff centricon, regardless of the elution methods. The concentrated sample was then injected into a Superose 6 increase 10/300 GL column (Cytiva) pre-equilibrated with TBS supplemented with 1 mM GABA and other ligands. Selected fractions corresponding to the nα1-GABAAR–Fab complex were combined and concentrated to about 0.1 mg ml−1 using a centricon with a 50 kDa cutoff.
Mass spectroscopy protein identification
The protein mass spectroscopy analysis was carried out with native receptor samples (approximately 5 μg protein) as previously described54. The proteins identified are summarized in Extended Data Fig. 1 and provided in Supplementary Table 1.
The protein mass spectroscopy analysis was carried out with native receptor samples (approximately 5 μg protein) as previously described55, except that the canonical Uniprot56 protein sequences from Mus musculus were used during data analysis. Both isoforms of γ2 (short and long) were included to probe their presence with mass spectroscopy. Identified proteins and peptides are provided in Supplementary Tables 2 and 3, respectively.
Single-molecule photobleaching of nα1-GABAAR–Fab complexes
Coverslips and glass slides were extensively cleaned, passivated and coated with methoxy polyethylene glycol (mPEG) and 2% biotinylated PEG as previously described57. A flow chamber was created by drilling 0.75 mm holes in the glass slide and placing double-sided tape between the holes. A coverslip was placed on top of the slide, and the edges were sealed with epoxy, creating small flow chambers. A concentration of 0.25 mg ml−1 streptavidin was then applied to the slide, incubated for 5 min and washed off with buffer consisting of 50 mM Tris, 50 mM NaCl and 0.25 mg ml−1 bovine serum albumin (BSA), pH 8.0. Anti-GFP nanobody (plasmid of the GFP nanobody was a gift from B. Collins) was expressed and purified according to the published protocol58. Biotinylation was carried out with a maleimide-PEG2-Biotin kit (ThermoFisher). Biotinylated nanobody at 7.5 µg ml−1 was applied to the slide, incubated for 10 min and washed off with 30 µl buffer A (20 mM Tris, 150 mM NaCl, pH 8) supplemented with 0.2 mg ml−1 BSA. nα1-GABAAR–Fab complexes in nanodiscs were eluted from the streptactin-XT resin, with biotin instead of 3C protease cleavage to preserve the GFP moiety. The sample was further purified by fluorescence-detection size-exclusion chromatography, and the peak corresponding to the complex was hand collected, which separated the native receptor from free Fab. The sample was diluted 1:30 to about 50 pM on the basis of fluorescence quantitation, applied to the chamber and incubated for 5 min before being washed off with 30 µl of buffer A. The chamber was immediately imaged using a Leica DMi8 TIRF microscope with an oil-immersion ×100 objective. Images were captured using a back-illuminated EMCCD camera (Andor iXon Ultra 888) with a 133 µm × 133 µm imaging area and a 13 µm pixel size. This 13 µm pixel size corresponds to 130 nm on the sample due to the ×100 objective.
Photobleaching movies were acquired by exposing the imaging area for 180 s. Single-molecule fluorescence time traces of nα1-GABAAR–Fab were generated using a custom Python script. Each trace was manually scored as having one to three bleaching steps, or was discarded if no clean bleaching steps could be identified. A total of about 450 molecules were evaluated from three separate movies. Scoring was verified by assessing the intensity of the spot; on average, the molecules that bleached in two steps were twice as bright as those that bleached in one step.
Scintillation proximation assay
YSI copper SPA beads from PerkinElmer were used to capture the nα1-GABAAR in the nanodisc via the MSP His-tag. Tritiated flunitrazepam from PerkinElmer was used as the radioligand, and clorazepate was used as the competing ligand to estimate background. During the ligand-binding assay setup, nα1-GABAAR in the nanodisc was first mixed with SPA beads and radioligand (2× beads), whereas the ligand of different concentrations (2× ligand) and competing ligand (2× background) were prepared using serial dilution. Then, an equal volume of 2× bead was mixed with 2× ligand (in triplicate) or 2× background in a 96-well plate. The final concentrations were 0.5 mg ml−1 SPA beads, approximately 1 nM native receptors, 10 nM [3H]flunitrazepam and 0.5 mM clorazepate in the background wells only. The plate was then read with a MicroBeta TriLux after a 2 h incubation. Specific counts were then imported into GraphPad Prism and analysed using a one-site competition model.
Negative-stain electron microscopy
Purified nα1-GABAAR–Fab complex in nanodiscs was first diluted with TBS to a concentration of approximately 0.05 mg ml−1. Continuous carbon grids (Ted Pella, catalogue no. 01844-F) were glow discharged with a PELCO easiGlow unit (Ted Pella) for 60 s at a current of 15 mA. A protein sample (5 μl) was applied to the carbon side of the grid held with a fine-tip tweezer and incubated for 10–30 s. The excessive sample was then wicked away from the side with a small piece of filter paper. The grid was quickly washed with 5 μl deionized water, followed by side wicking, which was repeated a total of three times. Immediately afterwards the grid was incubated with 5 μl 0.75% uranium formate for 45 s, wicked several times from the side and dried for at least 2 min at room temperature.
Cryo-EM sample preparation and data acquisition
We used a specific setup to streamline the preparation grids under different buffer and ligand conditions. First, buffers containing 10× ligand or additive were prepared and dispensed in 0.5 μl aliquots into a strip of 200 μl microcentrifuge tubes. Then, 5 μl purified nα1-GABAAR–Fab complex was added and quickly mixed by pipetting. Within 10 s, a 2.5 μl sample was applied to a glow-discharged (30 s at 15 mA) 200 mesh gold Quantifoil 2/1 grid overlaid with 2 nm continuous carbon (Ted Pella, catalogue no. 661-200-AU) and incubated for 30 s. The grid was blotted with a Mark IV Vitrobot under 100% humidity at 16 °C and flash frozen in liquid ethane. For the DID sample, no GABA was included during the purification, and the DID (2 μM) was added before vitrification. For the ZOL sample, 1 mM GABA was included throughout the purification, and 5 μM ZOL was added before vitrification using the above-mentioned PCR tube method. For the APG sample, 1 mM GABA and 5 μM APG were included from the membrane solubilization to the final size-exclusion chromatography.
Cryo-EM data were collected on a 300 keV Titan Krios equipped with a BioQuantum energy filter at either Pacific Northwest Cryo-EM Center or the Janelia cryo-EM facility. Data acquisition was automated using serialEM: defoci ranged between 0.9 to 2.5 μm, holes with suitable ice thickness were selected with the hole finder and combined to produce multishot–multihole targets, which enabled the acquisition of six movies per hole in each of the neighbouring nine holes. These movies were captured with a K3 direct electron detector. A total dose of 50 e−/Å2 was fractionated into 40 frames, with a dose rate of about 15 electrons per pixel per second for non-CDS mode or 7 electrons per pixel per second for CDS mode (Extended Data Table 1).
Cryo-EM data analysis
Super-resolution movies were imported to cryoSPARC59 v.3.3.1 and motion corrected using patch motion correction in cryoSPARC, with the output Fourier cropping factor set to ½. Initial contrast transfer function (CTF) parameters were then calculated using the patch CTF estimation in cryoSPARC. For each dataset, 2D class averages of particles picked by glob picker from approximately 1,000 micrographs were used as templates for the template picker. One round of 2D classification and several rounds of heterogeneous refinement seeded with ab initio models generated within cryoSPARC were used to select GABAAR particles, ranging from 4 to 6 million particles for our datasets. A non-uniform refinement (NU-Refinement) was performed to align these particles to a consensus structure. Two downstream strategies were used for our datasets (strategy 1 for the DID dataset, strategy 2 for the ZOL/GABA and the APG/GABA datasets), as described below.
Data processing strategy 1
Bin1 GABAAR particles, both images (360 × 360) and the star file converted using pyem60, were ported into RELION61 v.3.1. Then, a 3D autorefinement job with local search (angular sampling of 1.8°) was carried out to fine tune the particle poses in RELION. The refined structure, similar to that generated by cryoSPARC, had relatively weaker γ subunit transmembrane helices, which was reported previously10. To tackle this issue, we prepared a nanodisc mask in Chimera62 and carried out 3D classification without alignment (15 classes, regularization parameter T = 20) using that mask. The 3D classification can robustly give classes with much stronger transmembrane helices of the γ subunit. Those selected particles were imported into cryoSPARC and further refined using NU-Refinement with both defocus refinement and per-group CTF refinement options turned on. The consensus structure was a two-Fab bound structure, but earlier data processing revealed the presence of one-Fab species. Therefore, a 3D classification job was used with a mask focusing on the two binding sites of 8E3 Fab to isolate the one-Fab species. The one-Fab and two-Fab particles were separately refined with NU-Refinement and further refined with local refinement.
Data processing strategy 2
In this strategy the heterogeneity in Fab binding was addressed upstream in the data processing pipeline. As for strategy 1, GABAAR particles, at bin3 or 120 × 120, were imported into RELION for focused 3D classification. A reverse mask was prepared in cryoSPARC, which only excluded the TMD, to enable Fab binding at all possible positions. The 3D classification (10 classes, T = 20) gave clear two-Fab and one-Fab classes, and classes with incomplete Fab. Further 3D classification on these incomplete Fab particles produced only incomplete Fab classes, which led us to believe they were damaged particles and should be excluded from downstream processing. The two-Fab particles and the one-Fab particles, on the other hand, were imported into cryoSPARC, re-extracted at bin1 and separately refined with NU-Refinement. We still saw weak transmembrane helices for the γ subunit for the one-Fab and the two-Fab populations. To tackle this issue, instead of the 3D classification in RELION, we used the 3D classification (beta) job in cryoSPARC with a nanodisc mask, which was less robust but faster. Classes with stronger transmembrane helices were then combined and refined with NU-Refinement and finished with local refinement.
Global sharpening worked suboptimally for our nα1-GABAAR structures because of the local resolution variation and the lower signal-to-noise ratio for the TMD. The best method to sharpen our maps was achieved with LocScale63, which was used to represent some of our structures in Fig. 1. DeepEMhancer64 can yield comparable sharpening for the protein but not for the annulus lipids.
Subunit identification, model building, refinement and validation
Due to the subunit specificity of 8E3 Fab, the subunit with 8E3 Fab bound is defined as α1. The remaining subunits can be easily classified as α, β or γ from the characteristic N-linked glycosylation patterns of each subunit. It was clear that all 3D classes obtained were α-β-α-β-γ clockwise when viewed from the extracellular side of the membrane. Given the relative subunit abundance from earlier studies, we used α1-β2-α1-β2-γ2 as the starting model of the two-Fab class. We then examined the cryo-EM density maps to test our assignment in the context of sequence information. Specifically, we carefully examined residues for which the side chain can be unambiguously assigned and in which there is a difference of more than three carbon atoms or one sulfur atom between respective residues of the different receptor subunits. Regarding the non-α1 α subunit in the one-Fab classes, we further limited our scrutiny to positions showing no notable conformational differences in the corresponding two-Fab structure, to ensure the observed density difference was caused by the chemical identity of underlying residues.
For each dataset, the two-Fab bound nα1-GABAAR model was built first. The starting structures used were AlphaFold65 models of mouse GABAAR subunits and the best 8E3 Fab model generated with Rosetta66. These individual chains were first docked into the unsharpened cryo-EM density maps using the ‘fit-in-map’ tool of Chimera to assemble the full receptor–Fab complex. The full complex was then edited to remove unresolved portions and refined extensively to achieve better model–map agreement in Coot67. N-glycosylation was modelled using the carbohydrate module in Coot. Lipid and lipid-like molecules, including POPC, PIP2, dodecane and octane, were modelled using the CCP4 monomer library. New ligands included in this study, including their optimized geometry and constraint, were generated using phenix.elbow68. After the initial modelling, multiple runs of phenix.real_space_refinement69 and editing in Coot were carried out to improve the model quality.
The optimized two-Fab GABAAR structure was used as the starting model for one-Fab GABAAR structures. Although the one-Fab population probably consists of a mixture of α2/3 subunits at the α positions and a mixture of β1/β2 subunits at the β positions, we decided to use the α3 subunit and the β2 subunit for the modelling and subsequent structural comparisons, on the basis of our best interpretations of the density maps. We emphasize, nevertheless, that both α2 and β1 models reasonably fit the density maps. Shown in Extended Data Fig. 6 are sequence relationships between the subunits at chosen regions. The two-Fab structure was first docked into the one-Fab cryo-EM map using the ‘fit-in-map’ tool of ChimeraX70. Then, the aligned structure was edited in Coot to remove the extra Fab, replaced and renumbered the α1 sequence with the α3 sequence. This edited structure was further fitted and refined in Coot, first with secondary structure restraints generated with ProSMART71, and then without the restraints. Furthermore, certain residues and lipids were removed due to less clear density, and the glycosylation trees were remodelled. Similarly, this initial model was subjected to multiple runs of phenix.real_space_refinement and editing in Coot.
Animal use statement
Mouse carcasses donated from other laboratories of the Vollum Institute were used to establish and optimize the native GABAA receptor isolation workflow. The quantity of purified native receptor from each mouse was estimated using the fluorescence from the recombinant antibody fragment, which was then extrapolated to give the minimum number required for cryo-EM and biochemical analysis. For each native GABAA receptor preparation, 50 one-month-old (4–6 weeks) C57BL/6 mice (both male and female) were ordered from Charles River Laboratories. The housing conditions were set as: temperature 20–22 °C, humidity 40–60%, dark/light cycle 12:12 h. No randomization, blinding or experimental manipulations were performed on these animals. All mice were euthanized under the OHSU Institutional Animal Care and Use Committee (IACUC) protocols, consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA) and carried out only by members of the E.G. laboratory approved on IACUC protocol TR03_IP00000905.
Cell line statement
Sf9 cells for generation of baculovirus and expression of recombinant antibody fragment were from ThermoFisher (12659017, lot 421973). The cells were not authenticated experimentally for these studies. The cells were tested negative for mycoplasma contamination using the CELLshipper Mycoplasma Detection Kit M-100 from Bionique.
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