Cryo-EM structures of inactive and G_i-coupled GABA_B heterodimer

Abstract

22Metabotropic GABA B G protein-coupled receptor functions as a mandatory heterodimer of 23 GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. 24Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane 25 (TM) domain. Here we present cryo-EM structures of human full-length heterodimeric GABA B 26 receptor in the antagonist-bound inactive state and in the active state complexed with agonist 27 and positive allosteric modulator in the presence of G i1 protein at a resolution range of 2.8-3.0 28 Å. Cryo-EM analysis of the activated-GABA B -G i1 complex revealed that G i1 couples to the 29 activated receptor primarily in three major conformations, one via GB1 TM and two via GB2 30 TM, respectively. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, 31 which in turn triggers a rearrangement of TM interfaces between two subunits from TM3-32 TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the 33 opening of intracellular loop 3 and synergistically shifting of TM3, 4 and 5 helices in GB2 TM 34 domain to accommodate the α5-helix of G i1 . These results provide a structural framework for 35 understanding class C GPCR activation and a rational template for allosteric modulator design 36 targeting dimeric interface of GABA B receptor. 37 38 GABA B receptor belongs to the class C GPCR composed by metabotropic glutamate 54 receptors (mGlus), calcium-sensing receptor (CaSR) and taste 1 receptors 16 . Class C GPCR 55 form obligatory dimers, among them, mGlus and CaSR form the homodimer 17,18 , whereas 56 GABA B receptor is an obligatory heterodimer of two subunits GABA B1 (GB1) and GABA B2 57 (GB2) 19,20 . Each subunit is composed of a large extracellular 'Venus Flytrap' (VFT) domain, a 58 transmembrane (TM) domain, and a cytoplasmic tail 21 (Extended Data Fig. 1a, b). GB1 59 possesses an endoplasmic reticulum (ER) retention motif in its cytoplasmic tail 22 . The 60interaction between GB1 and GB2 facilitates its cell surface expression through coiled-coil 61 interactions in its cytoplasmic tail 23 . While GB1 is responsible for ligand recognition through 62 its VFT 24 , GB2 couples G i/o proteins through its TM 25,26 . However, a line of evidence show that 63 in the absence of GB2, GB1 asa (a GB1 mutant with deletion of ER retention signal) at the cell 64 surface or ER-localized GB1 also induces downstream signaling through G i/o proteins 27,28 . 65The crystal structures of isolated heterodimeric VFT of GABA B receptor in presence of the 66 antagonist or agonist revealed the open or closed conformation of GB1 VFT 29 . Structures of 67 isolated VFT and TM domains from other class C GPCR have been reported [30][31][32] , in addition to 68 more recently breakthrough that cryo-EM structures of full-length mGlu5 in apo state and in 69 the presence of agonist have been determined both at overall 4 Å resolution, providing the first 70 insights into the architecture of mGlu5 and structural framew...

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

“…Crystal structures of isolated mGluR1 (Wu et al, 2014) and mGluR5 (Dore et al, 2014, Christopher et al, 2019, Christopher et al, 2015 TMDs have shown TM1-mediated dimerization or monomers, respectively, while a cryogenic electron microscopy (cryo-EM) study of full-length mGluR5 (Koehl et al, 2019) showed no TMD interface in an inactive nanodisc-reconstituted form but a clear TM6 interface in a glutamate and positive allosteric modulator-bound pre-active state. Finally, a series of cryo-EM studies of the related GABA B receptors showed a lipid-mediated inactive TM5 interface that rearranged to a TM6 interface in the presence of agonist and a positive allosteric modulator (Mao et al, 2020, Papasergi-Scott et al, 2020, Park et al, 2020, Shaye et al, 2020. Together these studies point to a role for TMD rearrangement in mGluR activation, but the steps prior to formation of a putative active TM6 interface are not clear.…”

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors

“…However, the clear effect of perturbing this interface and the subtype differences suggest that it may be a potential target for specific and potent drugs. Intriguingly, inter-TMD PAM and NAM binding has recently been observed in cryo-EM structures of GABABRs (Mao et al, 2020, Shaye et al, 2020, indicating that TMD interfaces may be druggable sites across family C GPCRs.…”

“…Crystal structures of isolated mGluR1 (Wu et al, 2014) and mGluR5 (Dore et al, 2014) TMDs have shown TM1-mediated dimerization or monomers, respectively, while a cryogenic electron microscopy (cryo-EM) study of full-length mGluR5 (Koehl et al, 2019) showed no TMD interface in an inactive nanodisc-reconstituted form but a clear TM6 interface in a glutamate and positive allosteric modulator-bound pre-active state. Finally, a series of cryo-EM studies of the related GABAB receptors showed a lipid-mediated inactive TM5 interface that re-arranged to a TM6 interface in the presence of agonist and a positive allosteric modulator (Mao et al, 2020, Papasergi-Scott et al, 2020, Park et al, 2020, Shaye et al, 2020). Together these studies point to a role for TMD rearrangement in mGluR activation, but the steps prior to formation of a putative active TM6 interface are not clear.…”