Cryo-EM structures of ρ1 GABAA receptors with antagonist and agonist drugs

The family of ρ-type GABAA receptors includes potential therapeutic targets in several neurological conditions, and features distinctive pharmacology compared to other subtypes. Here we combine structures, recordings and simulations to characterize the binding and conformational impact of the drugs THIP (a non-opioid analgesic), CGP36742 (phosphinic acid inhibitor) and GABOB (an anticonvulsant) on a human ρ1 GABAA receptor. We identify a distinctive binding pose of THIP in ρ1 versus neuronal α4β3δ GABAA receptors, offering a rationale for its inverse effects on these subtypes. CGP36742 binding is similar to the canonical ρ-type inhibitor TPMPA, supporting a shared mechanism of action among phosphinic acid inhibitors. Binding of GABOB is similar to that of GABA, but produces a mixture of primed and desensitized states, likely underlying its weaker agonist activity. Together, these results elucidate interactions of a ρ-type GABAA receptor with therapeutic drugs, offering mechanistic insights and a prospective basis for further pharmaceutical development.


Introduction
γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the human central nervous system and it is crucial for the balance of neuronal excitation and inhibition 1 .Two types of GABA receptors have been identified based on their mechanism of action: GABA A receptors are GABA-gated chloride ion channels, while GABA B receptors are G protein-coupled receptors 1 .GABA A receptors belong to the pentameric ligand-gated ion channel (pLGIC) superfamily, which also contains ionotropic receptors for acetylcholine, glycine, and serotonin 2 .Channels in this family share a common architecture, with each of the five subunits contributing to an extracellular domain (ECD) with 10 strands (β1-β10) and a transmembrane domain (TMD) with 4 helices (M1-M4).The orthosteric ligand binding site is located at the subunit interface in the ECD, composed of loops A, B and C from the principal subunit and D, E and F from the complementary subunit.Ligand binding is thought to induce an anticlockwise rotation of the ECD relative to the TMD when viewed from the extracellular side.
These conformational changes propagate to the TMD, causing the opening of the pore formed by the M2 helices along the central axis, to allow ions to transit the lipid bilayer 3 .In contrast, GABA B receptors mediate relatively slow and prolonged synaptic inhibition, with presynaptic GABA B receptors suppressing neurotransmitter release, and postsynaptic GABA B receptors causing hyperpolarization of neurons 4 .
In humans, GABA A receptors are homo-or hetero-pentamers formed from a selection of 19 different subunits (α1-6, β1-3, γ1-3, ρ1-3, δ, ε, π and θ).Channels formed from ρ subunits were previously named GABA C receptors due to their distinctive physiological and pharmacological properties, including higher sensitivity to but slower activation by GABA relative to classical synaptic and extrasynaptic subtypes; this subtype is also relatively insensitive to bicuculline, barbiturates and benzodiazepines 5 , but sensitive to phosphinic acid compounds including (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) 6 , which provides possibilities to selectively modulate particular subforms of human GABA A receptors.Of the 3 types of ρ subunits found in mammals, ρ1 is expressed particularly in the retina, while ρ2 and ρ3 are widely distributed in brain 7,8 .ρ-type receptors play important roles in physiological processes including visual transduction 9 , postnatal neurodevelopment 10 , pain sensation 11 , and sleep-wake cycles 12 , and are potential therapeutic targets in myopia, sleep disorders, learning and memory disruption, peripheral nociception and anxiety 13,14 .
Several drugs act on ρ-type GABA A receptors, including the synthetic agents 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP, also known as gaboxadol) and 3-aminopropyl-n-butylphosphinic acid (CGP36742, also known as SGS742) and the natural product γ-amino-β-hydroxybutyric acid (GABOB, also known as buxamine) (Supplementary Fig. 1).THIP, a conformationally constrained derivative of muscimol, was developed as a non-opioid analgesic and antinociceptive agent 15,16 , and was a candidate in clinical trials for the treatment of insomnia 17 , Fragile X syndrome 18 and Angelman syndrome 19 .CGP36742 was the first GABA B receptor antagonist in clinical trials 20 , but is also an orally active antagonist of ρ-type GABA A receptors 21,22 , and has shown therapeutic potential for the treatment of cognitive deficits 23,24 .As a metabolite of GABA, GABOB is found endogenously in the mammalian central nervous system, but it is also an anticonvulsant used in the treatment of epilepsy 25 .The hydroxyl group at the C3 position of GABOB generates a stereogenic center, resulting in R and S enantiomeric forms; although (R)-GABOB is a modestly more potent anticonvulsant, the compound is applied clinically as a racemic mixture 26 .Despite their therapeutic relevance, the structural foundations of these drugs' selectivity and other functional properties at ρ-type GABA A receptors remain unclear.
Here, combining structures, electrophysiology and molecular dynamics (MD) simulations, we characterize the binding and structural impact of THIP, CGP36742 and GABOB on human ρ1 GABA A receptors.We identify a distinctive binding pose of THIP in ρ1 versus the extrasynaptic neuronal α4β3δ GABA A receptors, offering a rationale for its inverse effects on these subtypes.CGP36742 binding is similar to that of TPMPA, detailing a shared mechanism of action among phosphinic acid inhibitors.In contrast, GABOB binding is similar to that of GABA, but under equivalent conditions produces a mixture of primed and desensitized states; its density is compatible with both enantiomeric forms, likely representing the racemic mixture.Together, these results elucidate detailed interactions of a ρ-type GABA A receptor with therapeutic drugs, offering mechanistic insights and a prospective basis for further drug development.

Distinct states of ρ1 GABA A receptors resolved with antagonist and agonist drugs
We implemented a modified human ρ1 GABA A receptor construct (ρ1-EM) with truncated loops in the N-terminus and intracellular domain and an inserted fluorescent protein to facilitate expression while largely preserving wild-type function 27 .In Xenopus laevis oocytes expressing ρ1-EM, THIP and CGP36742 antagonized GABA activation (Fig. 1a,b), consistent with previous studies of wild-type channels 28,29 .GABOB functioned as an agonist, albeit at ~10-fold higher concentrations than GABA (Fig. 1c), consistent with its relatively weaker activity 30 .
To gain insight into the binding and conformational changes associated with these drugs, we solved cryogenic electron microscopy (cryo-EM) structures of ρ1-EM in complex with THIP, CGP36742 and GABOB in saposin nanodiscs with polar brain lipids to resolutions 2.0-2.4Å (Fig. 1, Supplementary Fig. 2, 3, 4, Table 1).We observed non-protein densities corresponding to the expected drugs in the five extracellular orthosteric ligand-binding sites of each structure; the corresponding maps enabled us to unambiguously build models for both the protein and drugs (Fig. 1, Supplementary Fig. 5).
Consistent with their functional roles as competitive inhibitors, THIP-and CGP36742-bound structures of ρ1-EM adopted the resting-like state, nearly identical to our previously reported apo structure 27 (Table 2).In the presence of the agonist GABOB, one class (13% of resolved particles) corresponded to the desensitized state previously reported with GABA 27 (Table 2).In another class (87% of resolved particles), despite the presence of GABOB in the orthosteric site, the ECD was only modestly activated, and the pore remained closed.This structure, corresponding neither to the resting nor desensitized states, aligned well with a previous so-called primed state determined in the presence of the negative modulator 17β-estradiol (E2) 31 (Supplementary Fig. 4e,f) (Table 2).Notably, E2 binds in a pocket located at the ECD-TMD interface, where it apparently disrupts allosteric transitions induced by GABA binding.In contrast, we observed no ligand density at the equivalent interface in the GABOB-bound structures, indicating a distinct mechanism of primed-state stabilization.(e) Cryo-EM maps as in d, viewed from the membrane plane.Insets show the drugs with corresponding densities.

Distinctive binding poses implicated in subtype-dependent effects of THIP
In our cryo-EM structure, THIP was bound in the orthosteric site with its two heterocycles perpendicular to loop C (Fig. 2a).The pyridine ring faced the principal subunit, with its amino group buried in an aromatic cage involving residues Y219, Y262 and Y268.The isoxazole ring faced the complementary subunit, with its hydroxyl and amino groups making polar interactions with principal residues S264 and T265, as well as complementary residues R125 and S189 (Fig. 2a,b, Supplementary Fig. 6).
To visualize structural changes induced by antagonist versus agonist binding, we superimposed the resting-like THIP complex with our previous GABA-bound desensitized structure 27 (Fig. 2c,d).The amino and hydroxyl groups of THIP roughly aligned with those of GABA; however, the bulky heterocycles of THIP were relatively protruded toward loop C, obstructing its lockdown over the antagonist.Thus, similar to TPMPA 27 , THIP appears to sterically occlude local activating transitions in the ρ-type orthosteric site.
Whereas THIP is a competitive antagonist of ρ1 32 (Fig. 1a), it is a partial agonist of α1β3γ2 GABA A receptors associated with the postsynapse, and a super-agonist of α4β3δ GABA A receptors found extrasynaptically [33][34][35] .Consistent with this behavior, a recent cryo-EM structure of the α4β3δ subtype was reported with THIP in an apparent desensitized state, with ligands at both β3(+)-α4(-) and δ(+)-β3(-) interfaces 36 .At both these interface types, THIP adopts a pose distinct from that in the ρ1 resting state, with the heterocycles rotated nearly 90°to lie parallel to the plane of loop C (Fig. 2e,f).This THIP pose in α4β3δ GABA A receptors is compatible with more extensive lockdown of loop C than in ρ1, apparently enabling activation.We previously observed that loop C is extended by one residue in ρ1 relative to β GABA A -receptor subunits, and that truncating the amino acid at the tip of loop C (ΔS264) results in functional properties more similar to postsynaptic subtypes 27 .THIP effects were similarly decreased in the ΔS264 variant, but remained inhibitory (Supplementary Fig. 7a), indicating that factors other than loop C length are involved in subtype-specific modulation.Although the structural basis for the distinct binding pose is not entirely clear, several bulky groups in the ρ1 orthosteric site are substituted with smaller sidechains in β3, including Y262 (β3-F200) on the principal subunit and R125 (β3-Q64) and S189 (β3-G127) on the complementary subunit (Supplementary Fig. 6).

Structural basis for receptor specificity among phosphinic acid inhibitors
In our cryo-EM structure, CGP36742 adopted an elongated pose, with its aminopropyl tail forming a salt bridge with E217 and cation-π interactions with the aromatic cage on the principal subunit face.The central phosphinic acid group was wedged between loop C on the principal subunit (making polar interactions with S264 and T265) and residue R125 on the complementary subunit, with the butyl tail extending toward the complementary β5 strand (Fig. 3a,b).The complex was comparable to the apo and THIP structures (Table 2, Fig. 3c), and nearly superimposable with our previous TPMPA structure (Fig. 3d), including analogous interactions of the amino and phosphinic acid groups; the additional butyl tail only required an alternative rotamer of M177 in the β5 strand (Fig. 3c,d).As in the case of TPMPA, the bulky, electronegative phosphinic acid group appeared to prevent lockdown of loop C over the antagonist, indicating a shared mechanism as well as binding pose among phosphinic acid inhibitors (Fig. 3e,f).Given the potency of CGP36742 to inhibit GABA B as well as ρ1 GABA A receptors 29,37 , we also compared its potential interactions with these structurally distinct targets.A structure of the related phosphinic acid inhibitor CGP35348 was previously reported with the ECD of a human GABA B receptor, bound in the cleft between ligand-binding lobes (LB1 and LB2) of the GBR1 subunit 38 (Fig. 4a-c).The CGP36742 pose from our ρ1-EM structure could be aligned into this GABA B receptor binding pocket on its equivalent aminopropyl and phosphinic acid moieties without clashing with any surrounding residues (Fig. 4b).Interestingly, the amino group of CGP36742 was oriented roughly opposite to that of CGP35348 in its resolved site (Fig. 4d), indicating the rotational flexibility of these compounds could support their polymodal activities.
The most potent and selective ρ1 antagonist identified thus far, (4-aminocyclopenten-1-yl)-butylphosphinic acid ((S)-4-ACPBPA), shares the phosphinic acid and butyl groups of CGP36742 but has a conformationally restricted aminocyclopentenyl group in place of the flexible aminopropyl tail 39,40 (Supplementary Fig. 1).Aligning (S)-4-ACPBPA with CGP36742 in our ρ1-EM complex showed this ligand could be accommodated without modification (Fig. 4e).Conversely, aligning this compound into the GABA B receptor resulted in a clash of the amino group with residue W65, suggesting a molecular basis for receptor specificity (Fig. 4f).

Primed and desensitized states captured with the weak racemic agonist GABOB
As described above, our cryo-EM dataset collected with GABOB contained particles in both apparent primed and desensitized states.The primed state exhibited only a partial lockdown of loop C over the ligand, corresponding to a limited (1.8 Å) S264-Cα translation, and a subtle (1.2°) domain rotation relative to the resting state (Fig. 5a).Although the contribution of this primed state to the receptor gating cycle remains unclear, the TMD was superimposable with that of the resting state,  Consistent with our previous structure with GABA 27 , the desensitized state with GABOB exhibited further lockdown of loop C (4.3 Å S264-Cα translation) and rotation between the ECD and TMD (7.3°) relative to the primed state (Fig. 5b).Interestingly, although all structures in this work contained ligands in the extracellular orthosteric site, local resolution in the ECD was relatively higher in the GABOB-desensitized state; in resting and primed structures, resolution was roughly similar between the domains (Supplementary Fig. 4a-d), suggesting that channel activation is associated with both stabilization of the ECD and mobilization of the TMD.
Densities for GABOB were clearly resolved in both the primed and desensitized states (Fig. 6a,b).Although the C3 carbon of GABOB constitutes a stereocenter, both enantiomers are ρ1 agonists, with modestly greater potency for (R)-GABOB 30 .As in medical practice, we used a racemic mixture in our experiments, such that our cryo-EM densities likely contained both forms; indeed, either (R)-or (S)-GABOB could be modeled into the ligand density in either structure (Fig. 6a,b).The amino end was coordinated by an aromatic cage (Y219, Y262, Y268) as well as by E217 in the principal subunit, while the carboxylate end made electrostatic contacts with R125 and S189 in the complementary subunit (Fig. 6c-f).In all cases, T265 at the tip of loop C could make a hydrogen bond with the C3 hydroxyl, consistent with the demonstrated effect of this residue on potency of both GABOB forms 41 .To substantiate binding capacity for both entantiomers, we performed all-atom molecular dynamics (MD) simulations of both the primed-and desensitized-state structures with both (R)-and (S)-GABOB.In quadruplicate ≥400-ns simulations of each system, both enantiomers remained within 2 Å median root-mean-square deviation (RMSD) relative to their starting poses in both structures (Fig. 6g, Supplementary Fig. 8), consistent with weak enantiomeric specificity.(g) Mobility of (R)-and (S)-GABOB in MD simulations of ρ1-EM in the primed (left, purple) and desensitized states (right, blue), as quantified by ligand RMSD (Å).Violin plots represent probability densities from 4 independent simulation replicates, sampled every 0.4 ns for the first 400 ns of each replicate (n = 4000), with markers indicating median and extrema.

Discussion
Pharmaceutical targeting of ρ-type GABA A receptors holds promise for drug development in treating visual, sleep, learning and memory disorders 13,42 .Given the limited sensitivity of this subtype to classical GABA A receptor modulators such as benzodiazepines, barbiturates and general anesthetics, the development of such agents will likely require a detailed understanding of ρ-specific mechanisms including binding, activation and inhibition.However, the specific mechanisms also means such drugs could limit interactions on neuronal receptors.The structural, functional and computational work presented here uncovers the binding modes of three drugs in the orthosteric site, highlighting among other things the critical role of loop C lockdown in channel gating.
Our structures highlight the critical role for loop-C lockdown in initiating ρ1 activation.Agonists such as GABA enable substantial lockdown of loop C, resulting in compaction of the orthosteric site and rotation of the ECD relative to the TMD (Fig. 7a).Alongside this apparent activated-desensitized state, treatment with the weaker agonist GABOB promotes a subclass in a presumably intermediary primed state, similar to a structure previously reported with the inhibitor estradiol, and exhibiting only a modest lockdown of loop C relative to the apo form.It remains unclear whether the predominance of the primed state arises from partial occupancy of the relatively low-potency agonist GABOB, or its limited efficacy under cryo-EM conditions; or indeed, why a fully activated-open state of ρ1 remains inaccessible under cryo-EM conditions 27,31 .On the other hand, antagonists such as THIP and phosphinic acids clearly obstruct loop-C lockdown altogether, resulting in an expanded orthosteric site superimposable with that of the apo structure (Fig. 7b,c).Among phosphinic acid inhibitors, lockdown is obstructed by a consistent binding pose of CGP36742 and TPMPA, and likely of the subtype-specific agent (S)-4-ACPBPA.Conversely, cross-reactivity of CGP36742 with GABA B receptor is attributable at least in part to rotational flexibility around the amino group.Subtype-and agent-specific interactions, e.g. with T265 on loop C, suggest avenues for future structure-based drug design.For instance, we observed a distinctive binding pose for THIP in ρ1-EM compared to a previously reported complex with an α4β3δ GABA A receptor 36 , which may underlie its opposing effects in these subtypes.Interestingly, the THIP derivative aza-THIP is a more selective ρ-type antagonist, with activity comparable to THIP at ρ1 but negligible at heteromeric GABA A receptors 28 .The two molecules are identical except at one heavy atom in the 5-membered ring, substituting a pyrazole in aza-THIP for the isoxazolo group in THIP (Supplementary Fig. 1a).In structures with THIP, the substituted oxygen atom appears to accept a hydrogen bond from principal-subunit loop C (T265) in ρ1-EM, but from the complementary subunit (ɑ4-T163 or β3-Q64) in the α4β3δ GABA A receptor.The differential environments for this substituted atom in particular may account for discriminating activity between these two receptors.A hydrogen bond with loop-C T265 also appears to underlie potency of both enantiomeric forms of GABOB in ρ1, and may contribute to the stabilization of a primed state in the presence of this weaker agonist relative to GABA.Taken together, this work details receptor-specific binding interactions of both antagonists and agonists in the orthosteric site, offering potential insights into differential pharmacology across multiple receptor subtypes in the GABAergic system.(c) Proposed dependence of orthosteric-site expansion/compaction on ligand identity, from relatively large antagonists to small agonists.corresponding to 0.6725 or 0.6645 Å/px using the software EPU 3.5.0(Thermo Fisher Scientific).The total dose was ~46 e -/Å 2 and defocus range was -0.8 to -1.8 μm.

Cryo-EM data processing
Dose-fractionated images in super-resolution mode were internally gain-normalized and binned by 2 in EPU during data collection.Cryo-EM data processing was first done in Relion 3.1.4 44, including Motion correction, contrast transfer functions (CTF) estimation with CTFFIND 4.1 45 , automatic particle picking with topaz 0.2.5 46 , particle extraction, 2D classification, 3D classification, 3D refinement, CTF refinement and polishing.Briefly, two rounds of 2D classification were done to remove junk particles, 3D classification (3 classes) was used to analyze the structural heterogeneity.Particles from classes with protein features were centered and re-extracted, and were used for the 3D refinement with symmetry C5.Several rounds of CtfRefine and one round of polishing were executed to improve the resolution.
The shiny particles were imported into CryoSPARC v4.2.1 for further processing 47 , including 3D classification with the PCA mode, and Non-Uniform Refinement 48 .

Model building and refinement
Model building was started with rigid body fitting of the previously published resting (PDB ID 8OQ6), primed (PDB ID 8RH7) or desensitized (PDB ID 8RH8) state structure into the density.The models were manually checked and adjusted in Coot 0.9.5 49 , and chemicals, waters and lipids were also manually added.The resulting model was further optimized using real-space refinement in PHENIX 1.18.2 50and validated by MolProbity 51 .Crystallographic information files (cif) for ligands were generated from isomeric SMILES strings using Grade2 52 .

Structural analysis
Pore radius profiles were calculated using CHAP 0.9.1 53 .Structure figures were prepared using UCSF ChimeraX 1.3 54 .ECD rotation was calculated as the dihedral angle between a) the Cα COM of the ECD (residues 97-280) of one subunit, b) the equivalent ECD residues of all subunits, c) the TMD (residues 281-479) including all subunits, and d) the equivalent TMD residues of one subunit.

Expression in oocytes and electrophysiology
mRNA encoding the ρ1-EM GABA A receptor was produced by in-vitro transcription using the mMessage mMachine T7 Ultra transcription kit (Ambion) according to the manufacturer protocol.
For recordings, glass electrodes were pulled and filled with 3 M KCl to give a resistance of 0.5-1.5 MΩ and used to clamp the membrane potential of injected oocytes at -60 mV with an OC-725C voltage clamp (Warner Instruments).Oocytes were maintained under continuous perfusion with Ringer's solution (123 mM NaCl, 10 mM HEPES, 2 mM KCl, 2 mM MgSO4, 2 mM CaCl2 , pH 7.5) at a flow rate around 1.5 mL/min.Buffer exchange was accomplished by manually switching the inlet of the perfusion system to the appropriate buffer.For assessing GABOB efficacy, a gravity-fed perfusion system was used as previously described to improve kinetics of solution exchange.Currents were digitized at a sampling rate of 2 kHz and lowpass filtered at 10 Hz with an Axon CNS 1440A Digidata system controlled by pCLAMP 10 (Molecular Devices).

Molecular dynamics simulation
Atomic coordinates of the ρ1-GABOB complex with GABOB built as either of two enantiomers were used as starting models for MD simulations.Each subunit was split into two chains for simulation, due to unresolved residues in the M3-M4 loop in the experimental structure.The simulation systems were set up in CHARMM-GUI 55 .The protein was embedded into a bilayer mimicking brain-lipid composition with the top leaflet containing 155 POPC, 24 POPE, and 38 cholesterol molecules and the bottom leaflet containing 65 POPC, 115 POPE, 26 POPS, and 32 cholesterol molecules.The protein-lipid complex was subsequently solvated with TIP3P water and 150 mM NaCl.The CHARMM36m forcefield 56 was used to describe the protein.Parameters for (R)-and (S)-GABOB were generated using CGenFF 57 in CHARMM-GUI.Cation-π specific NBFIX parameters were used to maintain appropriate ligand-protein interactions in the aromatic cage in the orthosteric binding site 58 .
Simulations were performed using GROMACS 2022.5 59 at 300 K and 1 bar using the velocity-rescaling thermostat 60 and Parrinello-Rahman barostat 61 .The LINCS algorithm was used to constrain the length of all bonds involving hydrogens 62 , and the particle mesh Ewald method 63 was used to calculate long-range electrostatic interactions.The systems were energy minimized and then equilibrated for 20 ns, with the position restraints on the protein and neurosteroids gradually released.
Four replicates, each > 400 ns, were simulated for each system as final unrestrained production runs.Before analysis, MD simulation trajectories were aligned on the Cα atoms of the ECD using MDAnalysis 64 .Root-mean-square deviations (RMSD) of the non-hydrogen atoms of GABOB were calculated in VMD 65 , and data from the first 400 ns were combined and plotted as violin plots using Matplotlib 65 .

Figure 1 .
Figure 1.Functional and structural profiles of ρ1 with antagonist and agonist drugs (a-c) Sample traces from two-electrode voltage-clamp electrophysiology recordings of ρ1-EM expressed in Xenopus oocytes exposed to THIP, CGP36742 or GABOB.(d) Cryo-EM maps of human ρ1-EM in complex with THIP (left), CGP36742 (middle) or GABOB (right), viewed from the extracellular side.Maps are colored by assigned functional states as similar to resting (green), primed (purple), or desensitized (blue).Low-pass filtered maps are shown in transparency to reveal lower-resolution features including the nanodisc and M3-helix intracellular-domain extension (M3 ICD).

Figure 2 .
Figure 2. Distinctive binding pose of THIP in the ρ1 GABA A receptor (a) Zoom view of the THIP (tan) binding site in ρ1-EM (green).Residues interacting directly with THIP are shown as sticks and colored by heteroatom; residues from the complementary subunit face are denoted (-).(b) Schematic of ρ1-EM interactions with THIP, colored as in a. Hydrogen bonds are indicated as dashed lines, hydrophobic and aromatic interactions as lashes.(c-d) Superimposition of the ECD of THIP-bound (green) and GABA-bound (PDB ID 8OP9, gray) ρ1-EM structures.For clarity, only two subunits are shown, aligned on the complementary subunit.(e-f) Zoom views as in d of the superimposition of the THIP binding site in ρ1-EM (green) and the α4β3δ GABA A receptor (PDB ID 7QND, gray), focusing on the β3-ɑ4 or δ-β3 interfaces, respectively.Structures are aligned on the ECD of the complementary subunit.

Figure 3 .
Figure 3. Common mechanism of ρ1 GABA A receptor antagonism by phosphinic acid inhibitors (a) Zoom view of CGP36742 (tan) binding site in ρ1-EM (green), colored and labeled as in Fig.2a.(b) Schematic of ρ1-EM interactions with CGP36742.Hydrogen bonds and other electrostatic interactions are indicated as dashed lines, hydrophobic and aromatic interactions as lashes.(c) Zoom view of superimposed structures of ρ1-EM with CGP36742 (green) and THIP (gray).(d) Zoom view of superimposed structures of ρ1-EM with CGP36742 (green) and TPMPA (PDB ID 8OQ7, gray).(e-f) Superimposition of the ECD of CGP36742-bound (green) and GABA-bound (PDB ID 8OP9, gray) ρ1-EM structures.For clarity, only two subunits are shown, aligned on the complementary subunit.

Figure 4 .
Figure 4. Comparative interactions of GABA B and GABA A receptors with phosphinic acid inhibitors (a) Chemical structure of CGP35348 (above) and its binding site in a human GABA B receptor (PDB ID 4MR8, green, below).The ligand (gray) and its direct residue contacts are colored by heteroatom.(b) Chemical structure of CGP36742 (above), and its superimposition into the equivalent GABA B -receptor site as in a.The ligand pose is adopted from the structure reported here with ρ1-EM, aligned on the shared aminopropyl and phosphinic acid moieties of CGP35348.(c) Overview of the ECD of the human GABA B receptor as shown in (a).(d)Alignment of CGP36742 in ρ1-EM (tan) with CGP35348 in the GABA B receptor (gray) as implemented in (b), colored by heteroatom.(e) Alignment of (S)-4-ACPBPA (tan) into the CGP36742 site of ρ1-EM.For perspective, principal and complementary subunits of ρ1-EM are colored purple and pink respectively; the ligand and interacting residues are colored by heteroatom.(f) Alignment of (S)-4-ACPBPA (tan) into the CGP35348 of the GABA B receptor site (green) shown in (a).Dashed circle indicates prospective clash with residue W65.
consistent with it representing a pre-active intermediate between resting and open.The presence of a substantial primed class with GABOB may reflect the relatively low affinity and slow kinetics of this agonist, despite the application of a supersaturating concentration (4 mM) for >30 min prior to grid freezing.

Figure 5 .
Figure 5. Primed and desensitized states captured with GABOB (a) Superimposed structures of the GABOB-bound primed state (purple) with a previously reported resting state (green, PDB 8OQ6).Structures are aligned on the TMD; for clarity, all but one of the subunits are semi-transparent.(b) Superimposed structures of GABOB-bound primed state (purple) and desensitization state (blue).The structures are aligned with the TMD domain, and for clarity all but one of the subunits are transparent.

Figure 6 .
Figure 6.Comparable accommodation of GABOB enantiomers in the orthosteric site (a) Modeling of (R)-(tan) and (S)-GABOB (cyan) into ligand density in the primed state of ρ1-EM (purple), colored by heteroatom.(b) Modeling of (R)-and (S)-GABOB into ligand density in the desensitized state of ρ1-EM (blue), otherwise colored as in a. (c) Zoom view of (R)-and (S)-GABOB in the primed state of ρ1-EM, colored as in a.(d) Zoom view of (R)-and (S)-GABOB in the desensitized state of ρ1-EM, colored as in b.(e) Schematics of (R)-(left, tan) and (S)-GABOB (right, cyan) interactions with the primed state of ρ1-EM.(f) Schematics of (R)-(left, tan) and (S)-GABOB (right, cyan) interactions with the desensitized state of ρ1-EM.In E-F, hydrogen bonds and other electrostatic interactions are indicated as dashed lines, hydrophobic and aromatic interactions as lashes.

Figure 7 .
Figure 7. Proposed mechanisms of antagonist and agonist drugs (a) Cartoons of ρ1 showing progressive ECD rotation between resting (green), primed (purple) and activated (open or desensitized, blue) states.Loop C is highlighted, and GABA and ions are shown in circles (carbon, tan; amine, blue; carboxylate, red; chloride, green).(b) Cartoon zoom view of the orthosteric binding site.GABA is shown in circles as in a. Green and blue lines depict lockdown of loop C.