Structure of human GABAB receptor in an inactive state

Abstract

Human GABA B G protein-coupled receptor (GPCR), a member of the class C family, mediates inhibitory neurotransmission and is implicated in epilepsy, pain, and addiction 1 . A unique GPCR known to require heterodimerization for function 2 – 6 , its two subunits, GABA B1 and GABA B2 , are structurally homologous but perform distinct and complementary functions. GABA B1 recognizes orthosteric ligand 7 , 8 , while GABA B2 couples with G protein 9 – 14 . Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane (TM) domain, and a cytoplasmic tail 15 . Whereas the VFT heterodimer structure has been resolved 16 , the structure of the full-length receptor and its transmembrane signaling mechanism remain unknown. Here we present a near full-length structure of the GABA B receptor, captured in an inactive state via cryo-electron microscopy (EM). Our structure reveals multiple ligands pre-associated with the receptor, including two large endogenous phospholipids embedded within the TM domains to maintain receptor integrity and modulate receptor function. We also identify a novel heterodimer interface between TM helices 5 and 3 of both subunits, which serves as a signature of the inactive conformation. A unique ′intersubunit latch′ within this TM interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

The GABAB Receptor—Structure, Ligand Binding and Drug Development

“…Changes in the 7TM subunit interaction interface upon agonist activation of the GABA B -R have been quantified by cysteine cross-linking studies [26], and is also described in the in the newly released papers describing cryo-EM structures [6,7]. The 7TM domain interface interactions in the inactive state is formed by TM3 (GABA B1 )-TM5 (GABA B2 ) and TM5 (GABA B1 )-TM3 (GABA B2 ) interactions and that the interactions between the protomers are facilitated through ionic interactions stabilized by aromatic residues [6,9,26]. The distance between the backbones of TM5 in each monomer is 8 Å and thereby much shorter than the distance between the homodimers in the inactive mGlu5, which is 21 Å [6,44].…”

“…Mao et al further suggest that G-proteins may bind to one of the subunits at a time due to sterically hindrance, favoring the GABA B2 as this was the most frequent populated distribution found in processing the cryo-EM data [ 6 ]. The recent cryo-EM structures describe that the extracellular half of the 7TM region of both monomers are occupied by a phospholipid, in both the active and inactive conformations, at a site corresponding to the orthosteric binding sites in most family A GPCRs [ 6 , 8 , 9 ]. Moreover, a “lid” is formed over the lipids in each subunit by residues located in the ECL2 of GABA B1b , and all ECLs in GABA B2 including the linker [ 8 , 9 ].…”

“…The recent cryo-EM structures describe that the extracellular half of the 7TM region of both monomers are occupied by a phospholipid, in both the active and inactive conformations, at a site corresponding to the orthosteric binding sites in most family A GPCRs [ 6 , 8 , 9 ]. Moreover, a “lid” is formed over the lipids in each subunit by residues located in the ECL2 of GABA B1b , and all ECLs in GABA B2 including the linker [ 8 , 9 ]. The lipids are suggested to play a role in receptor activation and structural integrity as seen by mutational studies of interacting residues in GABA B1b , trying to displace the lipid tail and interfere with coordination of the head group [ 8 , 9 ].…”

“…The paper by Shaye and coworkers [ 7 ] describes the full-length inactive apo receptor, an active receptor conformation bound to the agonist SKF97541 and the PAM GS39783, and two intermediate agonist-bound receptor conformations. The latest papers by Papasergi-Scott et al and Park et al describes the cryo-EM structure of the full-length inactive receptor [ 8 , 9 ]. These structures may provide new opportunities for understanding receptor mechanisms and for drug discovery.…”

Structure and Dynamics of GPCRs in Lipid Membranes: Physical Principles and Experimental Approaches

“…Two classes of cholesterol binding sites for β 2 AR were found by NMR spectroscopy [ 139 ], while MD simulation studies indicated direct interactions between cholesterol and GPCRs, including A 2A AR and β 2 AR [ 117 , 135 , 140 , 141 ]. Concerning GPCRs overall, at least seven distinct cholesterol binding sites have been observed in crystal structures of different receptors [ 136 , 137 , 142 , 143 , 144 , 145 , 146 , 147 ] ( Table 1 and Figure 3 ). MD simulations also revealed seven potential cholesterol binding sites on the surface of β 2 AR.…”