Structural and functional dissection of reovirus capsid folding and assembly by the prefoldin-TRiC/CCT chaperone network.
Proceedings of the National Academy of Sciences of the United States of America
2021; 118 (11)
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.
View details for DOI 10.1073/pnas.2018127118
View details for PubMedID 33836586
View details for PubMedCentralID PMC7980406
Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6.
Heterotrimeric 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 composed of the GTP-bound transducin alpha subunit (GalphaT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis, leading to hyperpolarization of the photoreceptor cell. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GalphaT. The structure reveals two GalphaT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEgamma 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 GalphaT·GTP subunits and PDE6 that supports an alternating-site catalytic mechanism.
View details for DOI 10.1016/j.molcel.2020.09.013
View details for PubMedID 33007200
Structure of a Hallucinogen-Activated Gq-Coupled 5-HT2A Serotonin Receptor.
2020; 182 (6): 1574
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-HT2A 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 Galphaq 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-Galphaq 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.
View details for DOI 10.1016/j.cell.2020.08.024
View details for PubMedID 32946782
Structures of Gα Proteins in Complex with Their Chaperone Reveal Quality Control Mechanisms.
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.
View details for DOI 10.1016/j.celrep.2020.02.086
View details for PubMedID 32126208
Structures of metabotropic GABAB receptor.
GABA (γ-aminobutyric acid) stimulation of the metabotropic GABAB receptor results in prolonged inhibition of neurotransmission that is central to brain physiology1. GABAB belongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins2,3. GABAB, however, 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 show structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABAB extracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABAB subunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABAB dimers and have important implications for rational drug design targeting these receptors.
View details for DOI 10.1038/s41586-020-2469-4
View details for PubMedID 32580208
Vitamin E-based glycoside amphiphiles for membrane protein structural studies.
Organic & biomolecular chemistry
2018; 16 (14): 2489–98
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.
View details for PubMedID 29564464
Dendronic trimaltoside amphiphiles (DTMs) for membrane protein study
2017; 8 (12): 8315–24
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.
View details for PubMedID 29619178
View details for PubMedCentralID PMC5858085
Simultaneous lipid and content mixing assays for in vitro reconstitution studies of synaptic vesicle fusion.
2017; 12 (9): 2014–28
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.
View details for DOI 10.1038/nprot.2017.068
View details for PubMedID 28858288
View details for PubMedCentralID PMC6163043
Functional synergy between the Munc13 C-terminal C-1 and C-2 domains
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 Ca(2+)-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 Ca(2+) influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.
View details for DOI 10.7554/eLife.13696
View details for PubMedID 27213521
Conformational Plasticity in the Transsynaptic Neurexin-Cerebellin-Glutamate Receptor Adhesion Complex.
Structure (London, England : 1993)
2016; 24 (12): 2163–73
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.
View details for DOI 10.1016/j.str.2016.11.004
View details for PubMedID 27926833
View details for PubMedCentralID PMC5149402
Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution.
Nature structural & molecular biology
2015; 22 (7): 555-564
Rapid neurotransmitter release depends on the Ca(2+) sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.
View details for DOI 10.1038/nsmb.3035
View details for PubMedID 26030874
Prevalent mechanism of membrane bridging by synaptotagmin-1
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (34): E3243-E3252
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.
View details for DOI 10.1073/pnas.1310327110
View details for Web of Science ID 000323271400015
View details for PubMedID 23918375
Reconstitution of the Vital Functions of Munc18 and Munc13 in Neurotransmitter Release
2013; 339 (6118): 421-425
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.
View details for DOI 10.1126/science.1230473
View details for Web of Science ID 000313960700036
View details for PubMedID 23258414
Membrane Bridging and Hemifusion by Denaturated Munc18
2011; 6 (7)
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.
View details for DOI 10.1371/journal.pone.0022012
View details for Web of Science ID 000292632000046
View details for PubMedID 21765933
NMR Structure and Calcium-Binding Properties of the Tellurite Resistance Protein TerD from Klebsiella pneumoniae
JOURNAL OF MOLECULAR BIOLOGY
2011; 405 (5): 1188-1201
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.
View details for DOI 10.1016/j.jmb.2010.11.041
View details for Web of Science ID 000287340200006
View details for PubMedID 21112337