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Metabotropic γ-aminobutyric acid receptors (GABA B ) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis 1 . GABA B belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits 2 . Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1–GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA B . Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface. Cryo-electron microscopy structures of apo, agonist- and positive allosteric modulator-bound forms of the GB1–GB2 heterodimer of the metabotropic γ-aminobutyric acid (GABA) receptor shed light on the activation mechanism of this receptor.

Disulfide trapping and FRET studies define an agonist-induced conformational change in mGlu2 from inactive symmetric dimers with an interface at transmembrane domains (TMs) 4 and 5 to an active state with TM6s serving as the dimer interface. G protein–coupled receptors (GPCRs) are major players in cell communication. Although they form functional monomers, increasing evidence indicates that GPCR dimerization has a critical role in cooperative phenomena that are important for cell signal integration. However, the structural bases of these phenomena remain elusive. Here, using well-characterized receptor dimers, the metabotropic glutamate receptors (mGluRs), we show that structural changes at the dimer interface are linked to receptor activation. We demonstrate that the main dimer interface is formed by transmembrane α helix 4 (TM4) and TM5 in the inactive state and by TM6 in the active state. This major change in the dimer interface is required for receptor activity because locking the TM4-TM5 interface prevents activation by agonist, whereas locking the TM6 interface leads to a constitutively active receptor. These data provide important information on the activation mechanism of mGluRs and improve our understanding of the structural basis of the negative cooperativity observed in these GPCR dimers.

Stimulation of the metabotropic GABA receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology . GABA belongs to family C of the G-protein-coupled receptors, which operate as dimers to transform synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins . However, GABA is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits . Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABA receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABA has an essential role in relaying structural transitions by ordering the linker that connects the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of each of the subunits of GABA caps and interacts with the hydrophilic head of a phospholipid that occupies the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G proteins. These results provide a starting framework through which to decipher the mechanistic modes of signal transduction mediated by GABA dimers, and have important implications for rational drug design that targets these receptors.

The γ-aminobutyric acid (GABA) type B receptor (GABA -R) belongs to class C of the G-protein coupled receptors (GPCRs). Together with the GABA receptor, the receptor mediates the neurotransmission of GABA, the main inhibitory neurotransmitter in the central nervous system (CNS). In recent decades, the receptor has been extensively studied with the intention being to understand pathophysiological roles, structural mechanisms and develop drugs. The dysfunction of the receptor is linked to a broad variety of disorders, including anxiety, depression, alcohol addiction, memory and cancer. Despite extensive efforts, few compounds are known to target the receptor, and only the agonist baclofen is approved for clinical use. The receptor is a mandatory heterodimer of the GABA and GABA subunits, and each subunit is composed of an extracellular Venus Flytrap domain (VFT) and a transmembrane domain of seven α-helices (7TM domain). In this review, we briefly present the existing knowledge about the receptor structure, activation and compounds targeting the receptor, emphasizing the role of the receptor in previous and future drug design and discovery efforts.

Protein quinary interactions organize the cellular interior and its metabolism. Although the interactions stabilizing secondary, tertiary, and quaternary protein structure are well defined, details about the protein–matrix contacts that comprise quinary structure remain elusive. This gap exists because proteins function in the crowded cellular environment, but are traditionally studied in simple buffered solutions. We use NMR-detected H/D exchange to quantify quinary interactions between the B1 domain of protein G and the cytosol of Escherichia coli . We demonstrate that a surface mutation in this protein is 10-fold more destabilizing in cells than in buffer, a surprising result that firmly establishes the significance of quinary interactions. Remarkably, the energy involved in these interactions can be as large as the energies that stabilize specific protein complexes. These results will drive the critical task of implementing quinary structure into models for understanding the proteome. Significance Globular proteins perform many of the chemical reactions required for life. The prevailing model of globular protein structure, which is based on studies in dilute solutions, emphasizes the requirement for a well-packed hydrophobic interior, but minimizes the importance of the exterior, provided it is hydrophilic. We demonstrate that the exterior plays a significant role when a globular protein is studied under physiologically relevant conditions. By changing a surface residue we show that attractive interactions between the protein surface and the cytosol modulate the stability of the protein, even though the change has a negligible effect in dilute solution. Recognizing and quantifying such intracellular interactions will aid in understanding and manipulating the biological role of proteins.

GABA B receptor is a Class C G protein‐coupled receptor (GPCR) for γ‐aminobutyric acid (GABA), the principal inhibitory neurotransmitter. It forms an obligatory heterodimer consisting of two subunits, GB1 and GB2. Whether the activation mechanism of the GABA B receptor is conserved during evolution remains unknown. Here, the cryogenic electron microscopy (cryo‐EM) structures of the drosophila GABA B receptor in both antagonist‐bound inactive state and GABA‐bound active state in complex with G i protein are reported. The drosophila GABA B receptor exhibits an asymmetric activation, mirroring its human homolog. However, a larger inactive interface prevents drosophila GABA B receptor constitutive activity. Four key residues, which are not conserved in drosophila GABA B receptor, are responsible for the activity of the positive allosteric modulator in its human homolog. Whereas the intracellular loop 2 of drosophila GB2 (dGB2) is less involved, the ordered C terminus of dGB2 and its corresponding region in its human homolog are required for G protein coupling. These evolutionary variations provide a complete understanding of the activation mechanism of the GABA B receptor and new insights for future development of allosteric modulators for medication and insecticides.

The heterodimeric GABA receptor, composed of GB1 and GB2 subunits, is a metabotropic G protein-coupled receptor (GPCR) activated by the neurotransmitter GABA. GABA binds to the extracellular domain of GB1 to activate G proteins through GB2. Here we show that GABA receptors can be activated by mechanical forces, such as traction force and shear stress, in a GABA-independent manner. This GABA-independent mechano-activation of GABA receptor is mediated by a direct interaction between integrins and the extracellular domain of GB1, indicating that GABA receptor and integrin form a mechano-transduction complex. Mechanistically, shear stress promotes the binding of integrin to GB1 and induces an allosteric re-arrangement of GABA receptor transmembrane domains towards an active conformation, culminating in receptor activation. Furthermore, we demonstrate that shear stress-induced GABA receptor activation plays a crucial role in astrocyte remodeling. These findings reveal a role of GABA receptor in mechano-transduction, uncovering a ligand-independent activation mechanism for GPCRs.

Many extracellular receptors are organized into complexes that may have functional implications. A combination of snap-tag protein labeling technology with time-resolved fluorescence resonance energy transfer (FRET) provides a method for the systematic analysis of higher-order protein-protein interactions on the surface of living cells. Cell-surface proteins are important in cell-cell communication. They assemble into heterocomplexes that include different receptors and effectors. Elucidation and manipulation of such protein complexes offers new therapeutic possibilities. We describe a methodology combining time-resolved fluorescence resonance energy transfer (FRET) with snap-tag technology to quantitatively analyze protein-protein interactions at the surface of living cells, in a high throughput–compatible format. Using this approach, we examined whether G protein–coupled receptors (GPCRs) are monomers or assemble into dimers or larger oligomers—a matter of intense debate. We obtained evidence for the oligomeric state of both class A and class C GPCRs. We also observed different quaternary structure of GPCRs for the neurotransmitters glutamate and γ-aminobutyric acid (GABA): whereas metabotropic glutamate receptors assembled into strict dimers, the GABA B receptors spontaneously formed dimers of heterodimers, offering a way to modulate G-protein coupling efficacy. This approach will be useful in systematic analysis of cell-surface protein interaction in living cells.

Metabotropic GABA B receptor is a G protein-coupled receptor that mediates inhibitory neurotransmission in the CNS. It functions as an obligatory heterodimer of GABA B receptor 1 (GBR1) and GABA B receptor 2 (GBR2) subunits. The association between GBR1 and GBR2 masks an endoplasmic reticulum (ER) retention signal in the cytoplasmic region of GBR1 and facilitates cell surface expression of both subunits. Here, we present, to our knowledge, the first crystal structure of an intracellular coiled-coil heterodimer of human GABA B receptor. We found that polar interactions buried within the hydrophobic core determine the specificity of heterodimer pairing. Disruption of the hydrophobic coiled-coil interface with single mutations in either subunit impairs surface expression of GBR1, confirming that the coiled-coil interaction is required to inactivate the adjacent ER retention signal of GBR1. The coiled-coil assembly buries an internalization motif of GBR1 at the heterodimer interface. The ER retention signal of GBR1 is not part of the core coiled-coil structure, suggesting that it is sterically shielded by GBR2 upon heterodimer formation.

Metabotropic GABA B receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABA B receptor complexes contain the principal subunits GABA B1 and GABA B2 , which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABA B receptors and modify the kinetics of GABA B receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABA B receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABA B2 receptor. A single GABA B2 C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABA B2 –KCTD16 interface disrupted both the biochemical association and functional modulation of GABA B receptors and G protein-activated inwardly rectifying K + channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABA B receptors. Defining the binding interface of GABA B receptor and KCTD reveals a potential regulatory site for modulating GABA B -receptor function in the brain.