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Neurotransmitter release depends critically on Munc18-1, Munc13, the Ca 2+ sensor synaptotagmin-1, and the soluble N-ethylmaleimide—sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) syntaxin-1, synaptobrevin, and SNAP-25. In vitro reconstitutions have shown that syntaxin-1—SNAP-25 liposomes fuse efficiently with synaptobrevin liposomes in the presence of synaptotagmin-1—Ca 2+ , but neurotransmitter release also requires Munc18-1 and Munc13 in vivo. We found that Munc18-1 could displace SNAP-25 from syntaxin-1 and that fusion of syntaxin-1—Munc18-1 liposomes with synaptobrevin liposomes required Munc13, in addition to SNAP-25 and synaptotagmin-1-Ca 2+ . Moreover, when starting with syntaxin-1—SNAP-25 liposomes, NSF—α-SNAP disassembled the syntaxin-1—SNAP-25 heterodimers and abrogated fusion, which then required Munc18-1 and Munc13. We propose that fusion does not proceed through syntaxin-1—SNAP-25 heterodimers but starts with the syntaxin-1—Munc18-1 complex; Munc18-1 and Munc13 then orchestrate membrane fusion together with the SNAREs and synaptotagmin-1-Ca 2+ in an NSF- and SNAP-resistant manner.

Family C G-protein-coupled receptors (GPCRs) operate as obligate dimers with extracellular domains that recognize small ligands, leading to G-protein activation on the transmembrane (TM) domains of these receptors by an unknown mechanism 1 . Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric G i . Upon activation of the extracellular domain, the two transmembrane domains undergo extensive rearrangement in relative orientation to establish an asymmetric TM6–TM6 interface that promotes conformational changes in the cytoplasmic domain of one protomer. Nucleotide-bound G i can be observed pre-coupled to inactive mGlu2, but its transition to the nucleotide-free form seems to depend on establishing the active-state TM6–TM6 interface. In contrast to family A and B GPCRs, G-protein coupling does not involve the cytoplasmic opening of TM6 but is facilitated through the coordination of intracellular loops 2 and 3, as well as a critical contribution from the C terminus of the receptor. The findings highlight the synergy of global and local conformational transitions to facilitate a new mode of G-protein activation. Cryo-electron microscopy structures show that metabotropic glutamate receptor 2 forms a dimer to which only one G protein is coupled, revealing the basis for asymmetric signal transduction.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca 2+ , is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders 1 . CaSR is a family C G-protein-coupled receptor 2 that functions as an obligate homodimer, with each protomer composed of a Ca 2+ -binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca 2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling.

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by the presence of auto-proteolysing extracellular regions that are involved in cell–cell and cell–extracellular matrix interactions 1 . Self cleavage within the aGPCR auto-proteolysis-inducing (GAIN) domain produces two protomers—N-terminal and C-terminal fragments—that remain non-covalently attached after receptors reach the cell surface 1 . Upon dissociation of the N-terminal fragment, the C-terminus of the GAIN domain acts as a tethered agonist (TA) peptide to activate the seven-transmembrane domain with a mechanism that has been poorly understood 2 – 5 . Here we provide cryo-electron microscopy snapshots of two distinct members of the aGPCR family, GPR56 (also known as ADGRG1) and latrophilin 3 (LPHN3 (also known as ADGRL3)). Low-resolution maps of the receptors in their N-terminal fragment-bound state indicate that the GAIN domain projects flexibly towards the extracellular space, keeping the encrypted TA peptide away from the seven-transmembrane domain. High-resolution structures of GPR56 and LPHN3 in their active, G-protein-coupled states, reveal that after dissociation of the extracellular region, the decrypted TA peptides engage the seven-transmembrane domain core with a notable conservation of interactions that also involve extracellular loop 2. TA binding stabilizes breaks in the middle of transmembrane helices 6 and 7 that facilitate aGPCR coupling and activation of heterotrimeric G proteins. Collectively, these results enable us to propose a general model for aGPCR activation. Cryo-electron microscopy structures of GPR56 and latrophilin 3 show how the released tethered agonist peptide interacts with the transmembrane domain, suggesting a model for the activation mechanism of adhesion G-protein-coupled receptors.

The calcium-sensing receptor (CaSR) is a family C G-protein-coupled receptor 1 (GPCR) that has a central role in regulating systemic calcium homeostasis 2 , 3 . Here we use cryo-electron microscopy and functional assays to investigate the activation of human CaSR embedded in lipid nanodiscs and its coupling to functional G i versus G q proteins in the presence and absence of the calcimimetic drug cinacalcet. High-resolution structures show that both G i and G q drive additional conformational changes in the activated CaSR dimer to stabilize a more extensive asymmetric interface of the seven-transmembrane domain (7TM) that involves key protein–lipid interactions. Selective G i and G q coupling by the receptor is achieved through substantial rearrangements of intracellular loop 2 and the C terminus, which contribute differentially towards the binding of the two G-protein subtypes, resulting in distinct CaSR–G-protein interfaces. The structures also reveal that natural polyamines target multiple sites on CaSR to enhance receptor activation by zipping negatively charged regions between two protomers. Furthermore, we find that the amino acid l -tryptophan, a well-known ligand of CaSR extracellular domains, occupies the 7TM bundle of the G-protein-coupled protomer at the same location as cinacalcet and other allosteric modulators. Together, these results provide a framework for G-protein activation and selectivity by CaSR, as well as its allosteric modulation by endogenous and exogenous ligands. Cryo-electron microscopy structures of the human calcium-sensing receptor in complex with G i and G q proteins reveal how this receptor activates distinct G protein subtypes and how its function is modulated by a variety of ligands.

Synaptotagmin-1 functions as a Ca ²⁺ sensor in neurotransmitter release through its two C ₂ domains (the C ₂A and C ₂B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble N-ethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C ₂AB) was shown to bind simultaneously to two membranes via the Ca ²⁺-binding loops at the top of both domains and basic residues at the bottom of the C ₂B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC ₂AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC ₂AB oligomers bound to each membrane via the Ca ²⁺-binding loops, with no contact of the bottom of the C ₂B domain with the membranes. We now show that lnC ₂AB containing impurities indeed aggregates in solution, but properly purified lnC ₂AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC ₂AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C ₂B domain contacts the membrane in a sizeable population of molecules of both membrane-bound C ₂AB and membrane-bound lnC ₂AB. NMR data on nanodiscs show that a fraction of C ₂AB molecules bind to membranes with antiparallel orientations of the C ₂ domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C ₂AB and lnC ₂AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.

Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. Despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that the same single-chain camelid antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained structures of neurotensin 1 receptor bound to antagonist SR48692, μ-opioid receptor bound to alvimopan, apo somatostatin receptor 2 and histamine receptor 2 bound to famotidine. We expect this rapid, straightforward approach to facilitate the broad exploration of GPCR inactive states without the need for extensive engineering and crystallization. Cryo-EM has facilitated structural studies of membrane proteins, but inactive GPCRs have remained inaccessible due to their small size. Robertson et al. demonstrate a common nanobody-based approach to streamline the determination of such structures.

Stimulation of the metabotropic GABA B receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology 1 . GABA B 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 2 , 3 . However, GABA B is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits 4 , 5 . Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABA B receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABA B 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 B 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 B dimers, and have important implications for rational drug design that targets these receptors. Cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABA B receptors, combined with cellular signalling assays, shed light on the mechanisms that underpin signal transduction mediated by these receptors.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit 1 . To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR–G-protein complex. By monitoring the transitions of the stimulatory G s protein in complex with the β 2 -adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein–receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events. Time-resolved cryo-EM is used to capture structural transitions during G-protein activation stimulated by a G-protein-coupled receptor.

Drugs targeting the μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and β-arrestin recruitment. Cryo-EM structures of μOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and β-arrestins bind. These observations highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes. Cryo-EM structures of µ-opioid receptor complexes with two agonists coupled to molecular dynamics simulations and functional assays highlight distinct efficacy for G protein subtype activation and β-arrestin recruitment.