Cryo-EM structures of inactive and active GABAB receptor

Abstract

Metabotropic GABA_B G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABA_B receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of G_i1 protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of G_i1. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABA_B receptor.

Fig. 1. Cryo-EM structures of GABA_B receptor in the inactive and active states.

a, b Cryo-EM density maps (left), models (middle) and maps colored according to local resolution (right) for the GABA_B receptor in the presence of CGP54626 (slate; antagonist) (a) and in complex with baclofen (magenta; agonist), BHFF (steel blue; PAM) and G_i1 protein (b). The colored density map is a composite map generated with the VFT and TMD locally refined map. The cryo-EM density map before focused refinement in transparent superposed with the final map, illustrating density for detergent micelle and G_i1 protein. The local resolution (Å) was calculated with the locally refined map as input, indicating a range of 2.5–3.5 Å resolution in most map regions for both the inactive and active states. GB1 and GB2 in inactive state, blue and yellow, respectively; red and green in active state, respectively.

Fig. 2. Structural comparison of GABA_B receptor in inactive and active states.

a, b Orthogonal views of the superimposed structures of GABA_B receptor in inactive and active states, showing the domain repositioning upon agonist binding-induced activation. Side views (a) and intracellular views (b) of superposed structures are shown, with the active structure in translucent in the left panels and the inactive structure in translucent in the right panels, respectively. VFT domains and loops are omitted for clarity in b. Red arrows indicate the translation direction and distance for GB1 and GB2 (measured at extracellular tips of TM1 helices), respectively. Structures were aligned on the combined domains of GB1 VFT and GB2 lobe 1, the relatively stable parts of the receptor along activation pathway.

Fig. 3. Structural details of the TM heterodimeric interfaces for the inactive and active GABA_B structures.

a, b Side (a) and intracellular (b) views of the inactive TM interface. EM densities (magenta) corresponding to putative cholesterols are shown in surface. c Close view of inactive TM interface proximal to the cytoplasm. H689^3.55 and E790^5.60 of GB1, H579^3.55 and E677^5.60 of GB2 forms salt bridge network to stabilize an inactive conformation. d Detailed interactions of the TM interface in the active state. e, f Cryo-EM density (e) and the detailed interactions (f) of (+)-BHFF (PAM) in the active TM interface. The critical residues are shown as sticks. Hydrogen bonds are depicted as dashed lines.

Fig. 4. Structural comparison of individual TM domains between inactive and active states.

a, b GB1 TM domain (a) and GB2 TM domain (b) between inactive and active states are superposed, respectively. Side, extracellular and intracellular views are shown. Magnified views of the stalk domains are shown in the left. Red arrows represent the movement direction and distance of TMs and loops.

Fig. 5. Three distinct modes of G_i1 coupling to GABA_B receptor.

a–c Cryo-EM density maps and the models of the GABA_B–G_i1 complex in B2a state (a), B2b state (b), and B1 state (c). GB1 in red, GB2 in green, Gαi in gold, Gβ in cyan, Gγ in purple and scFv16 in gray. d Magnified view of the interaction between the α5-helix of the Gαi and GABA_B receptor in B2a state is shown. Intracellular loops 1, 2, and 3 are highlighted. e Superposition of the B1 state with either B2a state or B2b state showing the potential steric clash between Gi proteins, indicating that only one Gi protein can bind to GABA_B receptor at one time. Gi in B2a state, brown; Gi in B2b state, blue; Gi in B1 state, gray.