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Structural basis for GABAA receptor potentiation by neurosteroids

Nature Structural & Molecular Biology volume 24, pages 986–992 (2017)Cite this article

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Abstract

Type A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABAARs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABAAR construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABAAR potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABAARs, a process of broad relevance to pentameric ligand-gated ion channels.

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Figure 1: α5TMD forms a functional gating unit potentiated by pregnanolone at the Gln245 site.
Figure 2: Architecture of GABAAR α5TMD.
Figure 3: The neurosteroid potentiation site.
Figure 4: Computational docking of pregnanolone and related neurosteroids.
Figure 5: Pore conformation and neurosteroid-induced motions in α5TMD.
Figure 6: TMD motions relative to the ECD.

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Acknowledgements

We thank staff at Diamond Light Source beamlines I03 and I04 for assistance at the synchrotron, K. Harlos and T. Walter for technical support with crystallization, J. Kammonen and staff at Pfizer for very kind time and assistance with electrophysiology, E. Beke for technical assistance during nanobody discovery, Y. Zhao for technical support with tissue culture, members of STRUBI for helpful discussions and G. Sutton and T. Malinauskas for feedback regarding the manuscript. This work was supported by the UK Biotechnology and Biological Sciences Research Council grant BB/M024709/1 (A.R.A. and P.S.M.), the UK Medical Research Council grants MR/L009609/1 and MC_UP_1201/15 (A.R.A.), the Human Frontier Science Program grant RGP0065/2014 (A.R.A.) and the Wellcome Trust studentships 105247/Z/14/Z (S.S.) and 084655/Z/08/Z (S.M.). We also thank INSTRUCT, part of the European Strategy Forum on Research Infrastructures and the Research Foundation-Flanders (FWO) for their nanobody discovery support. Further support from the Wellcome Trust Core Award 090532/Z/09/Z is acknowledged.

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  1. Paul S Miller and Suzanne Scott: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Paul S Miller, Suzanne Scott, Simonas Masiulis, Luigi De Colibus & A Radu Aricescu

  2. Neurobiology Division, MRC Laboratory of Molecular Biology, Cambridge, UK

    Suzanne Scott, Simonas Masiulis & A Radu Aricescu

  3. Structural Biology Brussels, Vrije Universiteit Brussel (VUB), Brussels, Belgium

    Els Pardon & Jan Steyaert

  4. VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium

    Els Pardon & Jan Steyaert

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  1. Paul S Miller
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Contributions

Experimental work was performed by P.S.M. and S.S. (protein expression, purification, crystallization, electrophysiology), S.M. (protein expression, purification), L.D.C. (docking experiments), E.P. and J.S. (nanobody generation) and A.R.A. (crystallography). The manuscript was written by P.S.M. and A.R.A. with input from all coauthors.

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Correspondence to Paul S Miller or A Radu Aricescu.

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Integrated supplementary information

Supplementary Figure 1 Detergent solubilised α5TMD binds pregnanolone at the Q245 neurosteroid potentiation site.

(a) Gel filtration profiles of purified α5TMD samples at 100 nM, heated beforehand for 1 hr at a range of temperatures to determine the decay of the pentameric peak protein species (a variable peak of protein aggregate is observed at 0.8 ml). (b) Thermostability plots of the pentameric peak decay with increasing temperature for α5TMD and its Q245L and Q245W mutants. Data points are from single melting experiments. (c) and (d) Gel filtration profiles for α5TMD and its Q245W mutant, respectively, showing the pentamer peak after heating to a temperature sufficient to cause 70 % peak decay (73 °C for α5TMD; 77 °C for Q245W), and subsequent dose-dependent stabilisation of the protein by increasing concentrations of pregnanolone. Remarkably, increasing pregnanolone concentrations drive α5TMD stability very close to its no heating (4 °C) level. (e) Pregnanolone dose-response curves for thermostabilisation of 70 % decayed α5TMD and its Q245L and Q245W mutants. NOTE: The solubility limit of pregnanolone in this buffer is 30 μM. Each data point represents mean ± s.e.m. of n = 3 experiments, each data point being from separate aliquots of purified protein.

Supplementary Figure 2 Architecture of the α5TMD-Nb25 complex.

(a) Side-on view of pregnanolone-bound (blue stick representation, in dashed box) α5TMD structure. Nb25 molecules are shown in green spacefill representation. Principal (P) and complementary (C) sides of one inter-subunit interface are labelled. (b) Close-up of Nb25 bound across the P/C interface near the neurotransmitter site. Dashed lines denote subunit/nanobody boundaries. Nb25 is shown in green, with a yellow CDR3 loop. The α5TMD P face is coloured in blue, with a cyan loop-C. (c) Close-up of interactions between the α5TMD neurotransmitter-binding loop-C (cyan) and the antigen recognising CDR3 loop of Nb25 (yellow). The closed conformation of the loop-C mimics that of the agonist-bound GABAAR-β3 (beige overlay, PDB ID: 4COF) rather than the open loop-C conformation of the antagonist-bound glycine receptor (grey overlay, PDB ID: 5CFB). (d) Binding footprint of Nb25 peeled 180° away from α5TMD. The hypervariable Nb25 regions, CDR1 (dashed circle), CDR2 (double-bordered ellipse) and CDR3 (single bordered ellipse), map broadly over the α5TMD β6-7 loop, loop-F and loop-C, respectively. Salt-bridge contacts are shown in red (Asp/Glu) and purple (Arg/Lys), polar contacts in cyan and van der Waals contacts in orange. Each Nb25 molecule forms ~1080 Å2 interfaces with α5TMD, roughly equally distributed between P/C faces. (e) and (f) Structural alignment of α5TMD pregnanolone-bound (blue) with apo (grey) or GABAAR-β3 (beige) ECDs, respectively. RMSD values over 204 equivalent ECD Cα positions are 0.19 Å (pregnanolone-bound vs apo α5TMD) and 0.45 Å (pregnanolone-bound α5TMD vs GABAAR-β3). (g) Bar chart showing that the amount of potentiation of EC10 histamine (his) by 10 μM pregnanolone (preg) is the same in the presence or absence of 50 μM Nb25. Each data point represents mean ± s.e.m. of n = 5 experiments, each measurement being from different cells.

Supplementary Figure 3 Crystallographic quality control for pregnanolone binding to α5TMD.

(a) SigmaA-weighted 2Fo-Fc (blue, contoured at 2σ) and Fo-Fc (green/red contoured at +3σ/-3σ) electron density maps following refinement in the absence of pregnanolone. (b) The same electron density maps and contour levels following refinement in the presence of pregnanolone. (c) Final model, showing pregnanolone (cyan) bound between the principal (P) and complementary faces (C) of adjacent subunits (PDB chains C and B respectively). Thick dotted lines indicate contacts within hydrogen-bonding distance. Thin dotted lines mark the inter-subunit boundary. (d) Overlay of the five pregnanolone molecules in α5TMD (cyan as in (c), the other four, at equivalent inter-subunit sites, in green). Pregnanolone molecules were overlaid by superposing the complementary chains from each site. Putative hydrogen bond distances between the pregnanolone 3α-hydroxyl and the M1 Gln245 εO range from 2.7-2.8 Å. Putative hydrogen bond distances between the pregnanolone C20 ketone and the Thr309 hydroxyl range from 3.0-3.4 Å.

Supplementary Figure 4 Impact of residue mutations lining the pregnanolone binding site on potentiation, and location of α-subunit M4 residues Asn410 and Tyr413 outside the site.

(a) Dose response curves for pregnanolone potentiation of EC10 histamine responses for α5TMDT287K and with mutations to single residues lining the pregnanolone binding site. (b) and (c) Pregnanolone bound to the inter-subunit site between M3 residues on the principal face (indicated by P in box) and M1 residues on the complementary face (indicated by C in box). The complementary face is shown in (a) cartoon or (b) cartoon with overlaying space-fill to highlight the top border of the pocket (dashed line). Putative hydrogen bonds between pregnanolone and α5TMD residues are indicated by dotted lines. Two M4 residues, Asn410 and Tyr413 (green labels) that have previously been implicated in pregnanolone binding by mutagenesis studies are shown to reside outside the site. M1 Ile238 and M4 Phe406 (blue labels) close the top of the site and bisect between M1 Gln245 and M4 Asn410 and Tyr413. Each data point represents mean ± s.e.m. Pregnanolone EC50 n number: WT n = 5, Q245S n = 8, V246A n = 4, W249L n = 7, I305 n = 3, T309A n = 3. Each n EC50 measurement is from different cells.

Supplementary Figure 5 Pregnanolone binding mode and α5TMD potentiation by neurosteroids.

(a) Top panel: structural formula of pregnanolone. Lower panel: the neurosteroid binding site in the α5TMD structure with pregnanolone bound (blue sticks, shown within SigmaA-weighted 2Fo-Fc electron density map contoured at 1.3σ, grey mesh) overlaid with computationally docked pregnanolone (yellow sticks). (b) Dose response curves for pregnanolone (3α5β), allopregnanolone (3α5α) and epipregnanolone (3β5β) potentiation of EC10 histamine responses for α5TMDT287K. Each data point represents mean ± s.e.m. Steroid EC50 n number: WT n = 5, allopregnanolone n = 3, epipregnanolone n = 10. Each n EC50 measurement is from different cells.

Supplementary Figure 6 Comparison between pregnanolone-bound α5TMD and GABAAR-β3 channel pore profiles and hydrophobicity.

Cross-sections showing the pore-lining M2 helices and the solvent-accessible surface mapped and colored according to the Eisenberg hydrophobicity scale (Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R., J. Mol. Biol. 179, 125–142, 1984) (pale green is hydrophobic; bright green is polar). Panels a-c show α5TMD and panels d-f show GABAAR-β3 (PDB ID: 4COF). (a) and (d) Sagittal ‘slices’ along the longitudinal pore axis. The dashed lines mark the beginning of the views shown in c and f. (b-c) and (e-f) Top-down views of the pore. View points are from the dashed lines shown a and d for b and e, respectively, and from further down the pore, just above the 2’ ring, for c and f. The desensitisation gate of α5TMD has two constrictions, the hydrophobic 2’ Val260 ring (pale green) and the -2’ Pro256 ring. In GABAAR-β3, the desensitisation gate has a single constriction, formed by the -2’ Ala248 ring.

Supplementary Figure 7 Crystal packing of pregnanolone-bound and apo α5TMD.

(a) Pregnanolone-bound α5TMD (space group I23) and (b) apo α5TMD (space group C2). Ligands are not shown, for clarity. Coloring scheme: ECD – blue, TMD – red, Nb25 – green. Dashed boxed areas in the left-hand panels are enlarged as close-ups in the right-hand panels. The M1-M2 linker (yellow) is not involved in crystal contacts.

Supplementary Figure 8 Comparison between the α5TMD pregnanolone-binding site and GluCl TMD modulator site locations.

Top-down (top panel) and side-on (lower panel) views of two neighbouring subunit TMDs. (a) Pregnanolone-bound α5TMD. (b) Ivermectin-bound GluCl (PDB ID: 3RIF). Boxed “P” and “C” letters indicate principal and complementary faces, respectively. Whilst pregnanolone occupies a location in the lower half, and on the outer surface of the TMD, the ivermectin site is in the top half of the GluCl TMD and ivermectin inserts deeper in the intersubunit interfaces.

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Structure of the α5TMD pentamer and its pregnanolone binding sites.

Cartoon representation of the α5TMD pentamer, complexed with Nb25 (green) and pregnanolone (ball-and-stick and spacefilling models, carbon atoms in blue, oxygen atoms in red), followed by zoom-in on one neurosteroid potentiation site. Subunits each comprise the human GABAA β3 ECD (light/dark blue) and α5 TMD (M1–M4 -helices, red/pink). N-linked glycans are shown as spheres coloured by atom type (carbon atoms in orange, oxygen atoms in red). The overview structure and the inter-subunit location of pregnanolone molecules are highlighted by oscillations (0–16 s). The zoom-in on the neurosteroid potentiation site (17–27 s) shows pregnanolone bound to an inter-subunit site between M3 residues on the principal face of one subunit (red) and M1 residues on the complementary face of the next (pink). (MP4 22055 kb)

Structural impact of pregnanolone binding to α5TMD.

Overview of the structural transition between the TMDs of globally superposed apo (orange/light orange) and pregnanolonebound(red/pink) α5TMD. A close-up view of one neurosteroid site (0–8 s) reveals the reshaping taking place upon pregnanolone (balland-stick representation, carbon atoms in blue, oxygen atoms in red) binding. The upper portion of the site contracts between principal face Ile305 and complementary face Gln245, whereas the lower portion of the site expands between Ile305 and complementary face Trp249. This creates torque on the lower part of the M1 helix twisting it sideways and outwards. A top-down view of the TMDs (10–16 s) shows a sideways and outward flexing of the lower (intracellular) halves of the five subunit helical bundles through the transition from apo to pregnanolone-bound states. A top-down zoom-in view of the bottom of the pore (18–24 s) shows the flexing of the transmembrane helices, the swing of the M1–M2 linker, and the dilation of the lower ring, defined by Pro256, of the desensitisation gate (Val260 surrounds the upper ring). The M2 motions are further emphasised by a side-on view between two opposing M2 helices lining the pore (29–34 s), where residues forming the desensitisation gate are highlighted. (MP4 15779 kb)

Impact of pregnanolone binding on the intra-subunit ECD-TMD relative orientation.

Neurosteroid binding induces changes in the relative positions of ECDs vs TMDs. Cartoons are coloured in light/dark blue (ECD, both α5TMD structures) and orange/light orange (apo α5TMD TMD) or ruby/pink (pregnanolone-bound α5TMD TMD); one subunit is highlighted. Pregnanolone molecules are shown in ball-and-stick and space-filling representations, carbon atoms in blue, oxygen atoms in red. The movie oscillates between global superpositions of apo and pregnanolone-bound α5TMD pentamers viewed side-on (0–6s), followed by focus on a single subunit viewed from the outside of the pentamer (7–12 s) and subsequently rotated to look into the inter-subunit principal face (14–20 s). The change in shape of the helical bundle in response to preganolone binding is visible from both orientations and when viewed looking into the inter-subunit principal face (14–20 s) reveals the flexure along M2 and the outward swing of the lower half of the TMD helical bundle. These motions induce an overall straightening of individual subunits due to a rocking motion of the ECD and TMD about their shared interface. (MP4 5143 kb)

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Miller, P., Scott, S., Masiulis, S. et al. Structural basis for GABAA receptor potentiation by neurosteroids. Nat Struct Mol Biol 24, 986–992 (2017). https://doi.org/10.1038/nsmb.3484

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