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SummaryG protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating the exchange of guanine nucleotide in the Gα subunit. 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. Using variability analysis to monitor the transitions of the stimulatory Gs protein in complex with the β2-adrenergic receptor (β2AR) at short sequential time points after GTP addition, we identified the conformational trajectory underlying G protein activation and functional dissociation from the receptor. Twenty transition structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of events driving G protein activation upon 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 (MD) simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP upon closure of the alpha-helical domain (AHD) against the nucleotide-bound Ras-homology domain (RHD) correlates with irreversible 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 signaling events.

SUMMARY Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the Gα C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal α5 helix before allowing GTP-gated release of Gα from Ric-8A., Graphical Abstract, In Brief Seven et al. present cryoEM structures of Gα subunit folding intermediates in complex with their universal chaperone, Ric-8. Ric-8 forms key interactions with the Ras domain to prepare GTP-gated release of Gα subunits from their chaperone. The structures, complemented by biochemical and cellular experiments, reveal a folding quality control mechanism.

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.

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders1. CaSR is a family C G-protein-coupled receptor2 that functions as an obligate homodimer, with each protomer composed of a Ca2+-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 Ca2+ 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.

Drugs targeting the G protein-coupled μ-opioid receptor (μOR) are the most effective analgesics available but are also associated with fatal respiratory depression. While some partial opioid agonists appear to be safer than full agonists, the signaling pathways responsible for respiratory depression have yet to be elucidated. Here we investigated the structural and mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two μOR agonists with different safety profiles. LFT, one of the most potent and lethal opioids, and MP, a derivative from the kratom plant with reduced respiratory depression in animal studies at equianalgesic doses, exhibited markedly different signaling efficacy profiles for G protein subtype activation and recruitment of β-arrestins. Cryo-EM structures of the μOR-Gi1 complex with MP (2.5Å) and LFT (3.2Å) revealed that the two ligands engage distinct sub-pockets, and molecular dynamics (MD) simulations showed additional differences in the binding site that propagate to the intracellular side of the receptor where G proteins and β-arrestins bind. While MP favors the precise G protein-bound active state observed in the cryo-EM structures, LFT favors a distinct active state. These results highlight how drugs engaging different parts of the μOR orthosteric pocket can lead to distinct signaling outcomes.

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.

SummaryCryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. However, 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 different inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained the structure of human neurotensin 1 receptor (NTSR1) bound to antagonist SR48692, of µ-opioid receptor (MOR) bound to the clinical antagonist alvimopan, as well as the structures of the previously uncharacterized somatostatin receptor 2 (SSTR2) in the apo state and histamine receptor 2 (H2R) bound to the H2 blocker famotidine. Each of these structures yields novel insights into ligand binding and specificity. We expect this rapid, straightforward approach to facilitate the broad structural exploration of GPCR inactive states without the need for extensive engineering and crystallization.

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally

Serotonin (5-hydroxytryptamine [5-HT]) 5-HT2-family receptors represent essential targets for lysergic acid diethylamide (LSD) and all other psychedelic drugs. Although the primary psychedelic drug effects are mediated by the 5-HT

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 mechanism1. Here we show structures of homodimers of the family C metabotropic glutamate receptor 2 (mGlu2) in distinct functional states and in complex with heterotrimeric Gi. 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 Gi 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.

Intracellular protein homeostasis is maintained by a network of chaperones that function to fold proteins into their native conformation. The eukaryotic TRiC chaperonin (TCP1-ring complex, also called CCT for cytosolic chaperonin containing TCP1) facilitates folding of a subset of proteins with folding constraints such as complex topologies. To better understand the mechanism of TRiC folding, we investigated the biogenesis of an obligate TRiC substrate, the reovirus σ3 capsid protein. We discovered that the σ3 protein interacts with a network of chaperones, including TRiC and prefoldin. Using a combination of cryoelectron microscopy, cross-linking mass spectrometry, and biochemical approaches, we establish functions for TRiC and prefoldin in folding σ3 and promoting its assembly into higher-order oligomers. These studies illuminate the molecular dynamics of σ3 folding and establish a biological function for TRiC in virus assembly. In addition, our findings provide structural and functional insight into the mechanism by which TRiC and prefoldin participate in the assembly of protein complexes.

Hallucinogens like lysergic acid diethylamide (LSD), psilocybin, and substituted N-benzyl phenylalkylamines are widely used recreationally with psilocybin being considered as a therapeutic for many neuropsychiatric disorders including depression, anxiety, and substance abuse. How psychedelics mediate their actions—both therapeutic and hallucinogenic—are not understood, although activation of the 5-HT(2A) serotonin receptor (HTR2A) is key. To gain molecular insights into psychedelic actions, we determined the active-state structure of HTR2A bound to 25-CN-NBOH—a prototypical hallucinogen—in complex with an engineered Gαq heterotrimer by cryoelectron microscopy (cryo-EM). We also obtained the X-ray crystal structures of HTR2A complexed with the arrestin-biased ligand LSD or the inverse agonist methiothepin. Comparisons of these structures reveal determinants responsible for HTR2A-Gαq protein interactions as well as the conformational rearrangements involved in active-state transitions. Given the potential therapeutic actions of hallucinogens, these findings could accelerate the discovery of more selective drugs for the treatment of a variety of neuropsychiatric disorders.

Stimulation of the metabotropic GABAB receptor by γ-aminobutyric acid (GABA) results in prolonged inhibition of neurotransmission, which is central to brain physiology1. GABAB 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 proteins2,3. However, GABAB is unique in its function as an obligate heterodimer in which agonist binding and G-protein activation take place on distinct subunits4,5. Here we present cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signalling assays and atomistic simulations, these structures reveal that extracellular loop 2 (ECL2) of GABAB 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 GABAB 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 GABAB dimers, and have important implications for rational drug design that targets these receptors. Cryo-electron microscopy structures of heterodimeric and homodimeric full-length GABAB receptors, combined with cellular signalling assays, shed light on the mechanisms that underpin signal transduction mediated by these receptors.

SUMMARYHeterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is comprised of the GTP-bound transducin α subunit (GαT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis to transmit signals to the optic nerve. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GαT. The structure reveals two GαT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEγ subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GαT·GTP subunits and PDE6 that support an alternating-site catalytic mechanism.

Membrane proteins play critical roles in a variety of cellular processes. For a detailed molecular level understanding of their biological functions and roles in disease, it is necessary to extract them from the native membranes. While the amphipathic nature of these bio-macromolecules presents technical challenges, amphiphilic assistants such as detergents serve as useful tools for membrane protein structural and functional studies. Conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus it is essential to develop novel agents with enhanced properties. Here, we designed and characterized a novel class of amphiphiles with vitamin E (i.e., α-tocopherol) as the hydrophobic tail group and saccharide units as the hydrophilic head group. Designated vitamin E-based glycosides (VEGs), these agents were evaluated for their ability to solubilize and stabilize a set of membrane proteins. VEG representatives not only conferred markedly enhanced stability to a diverse range of membrane proteins compared to conventional detergents, but VEG-3 also showed notable efficacy toward stabilization and visualization of a membrane protein complex. In addition to hydrophile–lipophile balance (HLB) of detergent molecules, the chain length and molecular geometry of the detergent hydrophobic group seem key factors in determining detergent efficacy for membrane protein (complex) stability.

The critical contribution of membrane proteins in normal cellular function makes their detailed structure and functional analysis essential. Detergents, amphipathic agents with the ability to maintain membrane proteins in a soluble state in aqueous solution, have key roles in membrane protein manipulation. Structural and functional stability is a prerequisite for biophysical characterization. However, many conventional detergents are limited in their ability to stabilize membrane proteins, making development of novel detergents for membrane protein manipulation an important research area. The architecture of a detergent hydrophobic group, that directly interacts with the hydrophobic segment of membrane proteins, is a key factor in dictating their efficacy for both membrane protein solubilization and stabilization. In the current study, we developed two sets of maltoside-based detergents with four alkyl chains by introducing dendronic hydrophobic groups connected to a trimaltoside head group, designated dendronic trimaltosides (DTMs). Representative DTMs conferred enhanced stabilization to multiple membrane proteins compared to the benchmark conventional detergent, DDM. One DTM (i.e., DTM-A6) clearly outperformed DDM in stabilizing human β2 adrenergic receptor (β2AR) and its complex with Gs protein. A further evaluation of this DTM led to a clear visualization of β2AR-Gs complex via electron microscopic analysis. Thus, the current study not only provides novel detergent tools useful for membrane protein study, but also suggests that the dendronic architecture has a role in governing detergent efficacy for membrane protein stabilization.

This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca2+-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca2+ sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored. This protocol is based on a method developed to study yeast vacuolar fusion. In contrast to other protocols used to study the release machinery, this assay incorporates N-ethylmaleimide sensitive factor (NSF) and α-SNAP, which disassemble syntaxin-1 and SNAP-25 heterodimers. As a result, fusion requires Munc18-1, which binds to the released syntaxin-1, and Munc13-1, which, together with Munc18-1, orchestrates SNARE complex assembly. The protocol can be readily adapted to investigation of other types of intracellular membrane fusion by using appropriate alternative proteins. Total time required for one round of the assay is 4 d.

Synaptic specificity is a defining property of neural networks. In the cerebellum, synapses between parallel fiber neurons and Purkinje cells are specified by the simultaneous interactions of secreted protein cerebellin with pre-synaptic neurexin and post-synaptic delta-type glutamate receptors (GluD). Here, we determined the crystal structures of the trimeric C1q-like domain of rat cerebellin-1, and the first complete ectodomain of a GluD, rat GluD2. Cerebellin binds to the LNS6 domain of α- and β-neurexin-1 through a high-affinity interaction that involves its highly flexible N-terminal domain. In contrast, we show that the interaction of cerebellin with isolated GluD2 ectodomain is low affinity, which is not simply an outcome of lost avidity when compared with binding with a tetrameric full-length receptor. Rather, high-affinity capture of cerebellin by post-synaptic terminals is likely controlled by long-distance regulation within this transsynaptic complex. Altogether, our results suggest unusual conformational flexibility within all components of the complex.

Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca2+-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a ‘primed’ state that does not fuse but is ready for fast fusion upon Ca2+ influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.001, eLife digest In the brain, neurons communicate with each other using small molecules called neurotransmitters. Electrical signals in one neuron trigger the release of the neurotransmitters, which then bind to receptor proteins on another neuron nearby. Neurotransmitters are packaged into small compartments called synaptic vesicles and are released from the neuron when these vesicles fuse with the membrane that surrounds the cell. Many proteins are involved in regulating this process to ensure that neurotransmitters are released at the right place and time. A large protein called Munc13 plays an important role in the release of neurotransmitters. It contains many different regions, including a long domain called MUN and three additional domains called C1, C2B and C2C among others. However, it is not clear how all these domains work together to control neurotransmitter release. Here Liu, Seven et al. address this question using purified proteins inserted into membranes as well as experiments in neurons from mice. The experiments show that the C1, C2B and C2C domains all play key roles in neurotransmitter release. Together with the MUN domain, these three domains help to form bridges between synaptic vesicles and the membrane surrounding the neuron. These bridges could help other proteins involved in neurotransmitter release to form a group that induces vesicle fusion. Liu, Seven et al.’s findings also suggest that Munc13 proteins cooperate with other proteins to form a 'primed' state in which a synaptic vesicle is ready to rapidly fuse with a neuron’s membrane when triggered to do so by an electrical signal. A future challenge is to find out how the proteins that form this primed state promote vesicle fusion. DOI: http://dx.doi.org/10.7554/eLife.13696.002

Synaptotagmin-1 functions as a Ca(2+) sensor in neurotransmitter release through its two C2 domains (the C2A and C2B 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 (C2AB) 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 C2B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC2AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC2AB oligomers bound to each membrane via the Ca(2+)-binding loops, with no contact of the bottom of the C2B domain with the membranes. We now show that lnC2AB containing impurities indeed aggregates in solution, but properly purified lnC2AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC2AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C2B domain contacts the membrane in a sizeable population of molecules of both membrane-bound C2AB and membrane-bound lnC2AB. NMR data on nanodiscs show that a fraction of C2AB molecules bind to membranes with antiparallel orientations of the C2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C2AB and lnC2AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.

Reconstituting Synaptic Vesicle Fusion Membrane fusion reactions have been reconstituted in vitro, but often the reconstituted reactions have not directly mirrored the requirements for synaptic vesicle fusion in vivo. Previous work generally used only N -ethylmaleimide–sensitive factor (NSF) attachment protein SNAP receptors (SNAREs) and one or two additional components and could not explain why deletion of Munc18-1 or Munc13 abolishes neurotransmitter release completely, yielding the severe disruptions of synaptic vesicle release in knockout mouse. Ma et al. (p. 421 , published online 20 December; see the Perspective by Hughson ) now present a faithful reconstitution of synaptic vesicle fusion. Membrane fusion required Munc18-1 and Munc13 when the reconstitution experiments included all eight key components (three SNAREs, Munc18-1, Munc13, synaptotagmin-1, NSF, and α-SNAP).

Neuronal Munc18-1 and members of the Sec1/Munc18 (SM) protein family play a critical function(s) in intracellular membrane fusion together with SNARE proteins, but the mechanism of action of SM proteins remains highly enigmatic. During experiments designed to address this question employing a 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorescence de-quenching assay that is widely used to study lipid mixing between reconstituted proteoliposomes, we observed that Munc18-1 from squid (sMunc18-1) was able to increase the apparent NBD fluorescence emission intensity even in the absence of SNARE proteins. Fluorescence emission scans and dynamic light scattering experiments show that this phenomenon arises at least in part from increased light scattering due to sMunc18-1-induced liposome clustering. Nuclear magnetic resonance and circular dichroism data suggest that, although native sMunc18-1 does not bind significantly to lipids, sMunc18-1 denaturation at 37 °C leads to insertion into membranes. The liposome clustering activity of sMunc18-1 can thus be attributed to its ability to bridge two membranes upon (perhaps partial) denaturation; correspondingly, this activity is hindered by addition of glycerol. Cryo-electron microscopy shows that liposome clusters induced by sMunc18-1 include extended interfaces where the bilayers of two liposomes come into very close proximity, and clear hemifusion diaphragms. Although the physiological relevance of our results is uncertain, they emphasize the necessity of complementing fluorescence de-quenching assays with alternative experiments in studies of membrane fusion, as well as the importance of considering the potential effects of protein denaturation. In addition, our data suggest a novel mechanism of membrane hemifusion induced by amphipathic macromolecules that does not involve formation of a stalk intermediate.

The tellurium oxyanion TeO(3)(2-) has been used in the treatment of infectious diseases caused by mycobacteria. However, many pathogenic bacteria show tellurite resistance. Several tellurite resistance genes have been identified, and these genes mediate responses to diverse extracellular stimuli, but the mechanisms underlying their functions are unknown. To shed light on the function of KP-TerD, a 20.5 -kDa tellurite resistance protein from a plasmid of Klebsiella pneumoniae, we have determined its three-dimensional structure in solution using NMR spectroscopy. KP-TerD contains a β-sandwich formed by two five-stranded β-sheets and six short helices. The structure exhibits two negative clusters in loop regions on the top of the sandwich, suggesting that KP-TerD may bind metal ions. Indeed, thermal denaturation experiments monitored by circular dichroism and NMR studies reveal that KP-TerD binds Ca(2+). Inductively coupled plasma-optical emission spectroscopy shows that the binding ratio of KP-TerD to Ca(2+) is 1:2. EDTA (ethylenediaminetetraacetic acid) titrations of Ca(2+)-saturated KP-TerD monitored by one-dimensional NMR yield estimated dissociation constants of 18 and 200 nM for the two Ca(2+)-binding sites of KP-TerD. NMR structures incorporating two Ca(2+) ions define a novel bipartite Ca(2)(+)-binding motif that is predicted to be highly conserved in TerD proteins. Moreover, these Ca(2+)-binding sites are also predicted to be present in two additional tellurite resistance proteins, TerE and TerZ. These results suggest that some form of Ca(2+) signaling plays a crucial role in tellurite resistance and in other responses of bacteria to multiple external stimuli that depend on the Ter genes.

Neurotransmitter release depends critically on: the SNAREs syntaxin-1, synaptobrevin and SNAP-25, which form SNARE complexes that bridge the vesicle and plasma membranes; NSF/SNAPs, which disassemble SNARE complexes; Munc18-1, which binds to syntaxin-1 and, together with Munc13, orchestrates SNARE-complex assembly; and the Ca2+ sensor synaptotagmin-1. Previous attempts to reconstitute neurotransmitter release revealed efficient fusion of syntaxin-1/SNAP-25-liposomes with synaptobrevin-liposomes in the presence synaptotagmin-1/Ca2+, in stark contrast with physiological data showing that Munc18-1 and Munc13 are essential for neurotransmitter release. We now solve this paradox, showing that Munc18-1 displaces SNAP-25 from syntaxin-1 and that syntaxin-1/Munc18-1-liposomes fuse efficiently with synaptobrevin-liposomes in a manner that requires SNAP-25, Munc13-1 and synaptotagmin-1/Ca2+. Moreover, when starting with syntaxin-1/SNAP-25-liposomes, NSF/a-SNAP disassemble the syntaxin-1/SNAP-25 heterodimers, thus inhibiting fusion, and fusion then requires Munc18-1 and Munc13-1. These results suggest that, for the first time, our experiments reconstitute synaptic vesicle fusion with the eight major components of the release machinery. We propose a model whereby the pathway to synaptic vesicle fusion does not proceed through syntaxin-1/SNAP-25 heterodimers and starts at the syntaxin-1/Munc18-1 complex; Munc18-1 and Munc13 then orchestrate membrane fusion together with the SNAREs, synaptotagmin-1 and Ca2+ in a manner that is not inhibited by NSF/SNAPs.