RESUMO
Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
Assuntos
Microscopia Crioeletrônica/métodos , Lipídeos/química , Complexos Multiproteicos/química , Receptores de GABA-A/química , Imagem Individual de Molécula/métodos , Análise de Célula Única/métodos , Anticorpos de Domínio Único/química , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação ProteicaRESUMO
The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2; upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions that result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. In spite of several reported pLGIC structures4-8, no clear unifying view has emerged on the conformational transitions involved in channel gating. Here we report four cryo-electron microscopy structures of the full-length mouse 5-HT3 receptor in complex with the anti-emetic drug tropisetron, with serotonin, and with serotonin and a positive allosteric modulator, at resolutions ranging from 3.2 Å to 4.5 Å. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures include an 'open' state and two ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles, and characterize movements at the gate level and cation accessibility in the pore. Together, these data deepen our understanding of the gating mechanism of pLGICs and capture ligand binding in unprecedented detail.
Assuntos
Microscopia Crioeletrônica , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Animais , Sítios de Ligação , Ativação do Canal Iônico , Ligantes , Camundongos , Simulação de Dinâmica Molecular , Movimento/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/metabolismo , Serotonina/química , Serotonina/metabolismo , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Anticorpos de Domínio Único/farmacologia , Termodinâmica , Tropizetrona/química , Tropizetrona/metabolismo , Tropizetrona/farmacologiaRESUMO
5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors. SIGNIFICANCE STATEMENT: This review provides a comprehensive account of the classification and function of 5-hydroxytryptamine receptors, including how they are targeted for therapeutic benefit.
Assuntos
Farmacologia Clínica , Serotonina , Humanos , Ligantes , Receptores de SerotoninaRESUMO
Single-particle electron cryo-microscopy (cryo-EM) has become an effective and straightforward approach to determine the structure of membrane proteins. However, obtaining cryo-EM grids of sufficient quality for high-resolution structural analysis remains a major bottleneck. One of the difficulties arises from the presence of detergents, which often leads to a lack of control of the ice thickness. Amphipathic polymers such as amphipols (APols) are detergent substitutes, which have proven to be valuable tools for cryo-EM studies. In this work, we investigate the physico-chemical behavior of APol- and detergent-containing solutions and show a correlation with the properties of vitreous thin films in cryo-EM grids. This study provides new insight on the potential of APols, allowing a better control of ice thickness while limiting protein adsorption at the air-water interface, as shown with the full-length mouse serotonin 5-HT3A receptor whose structure has been solved in APol. These findings may speed up the process of grid optimization to obtain high-resolution structures of membrane proteins.
Assuntos
Detergentes , Tensoativos , Animais , Camundongos , Tensoativos/química , Microscopia Crioeletrônica , Elétrons , Gelo , Proteínas de MembranaRESUMO
Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Lipídeos/química , Animais , Ligantes , Mutagênese , XenopusRESUMO
Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.
Assuntos
Receptores 5-HT3 de Serotonina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurotransmissores/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores 5-HT3 de Serotonina/metabolismoRESUMO
General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.
Assuntos
Anestésicos Gerais/química , Anestésicos Gerais/metabolismo , Cianobactérias/química , Isoflurano/análogos & derivados , Canais Iônicos de Abertura Ativada por Ligante/química , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Propofol/química , Sítios de Ligação/genética , Cristalografia por Raios X , Desflurano , Fenômenos Eletrofisiológicos , Isoflurano/química , Isoflurano/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Propofol/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , PrótonsRESUMO
Pentameric ligand-gated ion channels from the Cys-loop family mediate fast chemo-electrical transduction, but the mechanisms of ion permeation and gating of these membrane proteins remain elusive. Here we present the X-ray structure at 2.9 A resolution of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC) at pH 4.6 in an apparently open conformation. This cationic channel is known to be permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constriction matches with rings of hydrophilic residues that are likely to contribute to the ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformation, shows a wider pore where the narrow hydrophobic constriction found in ELIC is removed. Comparative analysis of GLIC and ELIC reveals, in concert, a rotation of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation.
Assuntos
Cianobactérias/química , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Cristalografia por Raios X , Dickeya chrysanthemi/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismoRESUMO
GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.
Assuntos
Benzodiazepinas/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Benzodiazepinas/metabolismo , Sítios de Ligação , Biopolímeros , Cristalografia por Raios X , Canais Iônicos/química , Ligantes , Modelos Moleculares , Receptores de GABA-A/metabolismo , XenopusRESUMO
Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution.
Assuntos
Receptores 5-HT3 de Serotonina/isolamento & purificação , Animais , Cromatografia de Afinidade , Cromatografia em Gel , Cristalização , Eletroforese em Gel de Poliacrilamida , Glicosilação , Camundongos , Receptores 5-HT3 de Serotonina/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit ß1-ß2 and M2-M3 loops, with agonist binding re-positioning a key ß1-ß2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9' hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels.
Assuntos
Receptores Nicotínicos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Ativação do Canal Iônico/fisiologia , Conformação Molecular , Receptores Colinérgicos/metabolismo , Músculos/metabolismoRESUMO
Vortioxetine (VTX) is a recently approved antidepressant that targets a variety of serotonin receptors. Here, we investigate the drug's molecular mechanism of operation at the serotonin 5-HT3 receptor (5-HT3R), which features two properties: VTX acts differently on rodent and human 5-HT3R, and VTX appears to suppress any subsequent response to agonists. Using a combination of cryo-EM, electrophysiology, voltage-clamp fluorometry and molecular dynamics, we show that VTX stabilizes a resting inhibited state of the mouse 5-HT3R and an agonist-bound-like state of human 5-HT3R, in line with the functional profile of the drug. We report four human 5-HT3R structures and show that the human receptor transmembrane domain is intrinsically fragile. We also explain the lack of recovery after VTX administration via a membrane partition mechanism.
RESUMO
Recently discovered bacterial homologues of eukaryotic pentameric ligand-gated ion channels, such as the Gloeobacter violaceus receptor (GLIC), are increasingly used as structural and functional models of signal transduction in the nervous system. Here we present a one-microsecond-long molecular dynamics simulation of the GLIC channel pH stimulated gating mechanism. The crystal structure of GLIC obtained at acidic pH in an open-channel form is equilibrated in a membrane environment and then instantly set to neutral pH. The simulation shows a channel closure that rapidly takes place at the level of the hydrophobic furrow and a progressively increasing quaternary twist. Two major events are captured during the simulation. They are initiated by local but large fluctuations in the pore, taking place at the top of the M2 helix, followed by a global tertiary relaxation. The two-step transition of the first subunit starts within the first 50 ns of the simulation and is followed at 450 ns by its immediate neighbor in the pentamer, which proceeds with a similar scenario. This observation suggests a possible two-step domino-like tertiary mechanism that takes place between adjacent subunits. In addition, the dynamical properties of GLIC described here offer an interpretation of the paradoxical properties of a permeable A13'F mutant whose crystal structure determined at 3.15 A shows a pore too narrow to conduct ions.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cianobactérias/química , Cianobactérias/metabolismo , Ativação do Canal Iônico , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
The multifactorial origin and neurochemistry of Alzheimer's disease (AD) call for the development of multitarget treatment strategies. We report a first-in-class triple acting compound that targets serotonin type 6 and 3 receptors (5-HT-Rs) and monoamine oxidase type B (MAO-B) as an approach for treating AD. The key structural features required for MAO-B inhibition and 5-HT6R antagonism and interaction with 5-HT3R were determined using molecular dynamic simulations and cryo-electron microscopy, respectively. Bioavailable PZ-1922 reversed scopolamine-induced cognitive deficits in the novel object recognition test. Furthermore, it displayed superior pro-cognitive properties compared to intepirdine (a 5-HT6R antagonist) in the AD model, which involved intracerebroventricular injection of an oligomeric solution of amyloid-ß peptide (oAß) in the T-maze test in rats. PZ-1922, but not intepirdine, restored levels of biomarkers characteristic of the debilitating effects of oAß. These data support the potential of a multitarget approach involving the joint modulation of 5-HT6R/5-HT3R/MAO-B in AD.
Assuntos
Doença de Alzheimer , Serotonina , Ratos , Animais , Serotonina/efeitos adversos , Microscopia Crioeletrônica , Receptores de Serotonina , Antagonistas da Serotonina/farmacologia , Antagonistas da Serotonina/uso terapêutico , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/induzido quimicamente , Monoaminoxidase , Cognição , Inibidores da Monoaminoxidase/farmacologia , Inibidores da Monoaminoxidase/uso terapêuticoRESUMO
Fast synaptic communication requires receptors that respond to the presence of neurotransmitter by opening an ion channel across the post-synaptic membrane. The muscle-type nicotinic acetylcholine receptor from the electric fish, Torpedo, is the prototypic ligand-gated ion channel, yet the structural changes underlying channel activation remain undefined. Here we use cryo-EM to solve apo and agonist-bound structures of the Torpedo nicotinic receptor embedded in a lipid nanodisc. Using both a direct biochemical assay to define the conformational landscape and molecular dynamics simulations to assay flux through the pore, we correlate structures with functional states and elucidate the motions that lead to pore activation of a heteromeric nicotinic receptor. We highlight an underappreciated role for the complementary subunit in channel gating, establish the structural basis for the differential agonist affinities of α/δ versus α /γ sites, and explain why nicotine is less potent at muscle nicotinic receptors compared to neuronal ones.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante , Receptores Nicotínicos , Animais , Sítios de Ligação , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Ligantes , Músculos , Receptores Nicotínicos/metabolismo , Torpedo/metabolismoRESUMO
Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.
Assuntos
Neurotoxinas , Receptores Nicotínicos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Elapidae , Humanos , Músculos/metabolismo , Neurotoxinas/química , Receptores Nicotínicos/metabolismoRESUMO
Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.
Assuntos
Técnicas Biossensoriais , Canais Iônicos de Abertura Ativada por Ligante , Animais , Camundongos , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , LigantesRESUMO
Pentameric ligand-gated ion channels (pLGICs) are widely expressed in the animal kingdom and are key players of neurotransmission by acetylcholine (ACh), gamma-amminobutyric acid (GABA), glycine and serotonin. It is now established that this family has a prokaryotic origin, since more than 20 homologues have been discovered in bacteria. In particular, the GLIC homologue displays a ligand-gated ion channel function and is activated by protons. The prokaryotic origin of these membrane proteins facilitated the X-ray structural resolution of the first members of this family. ELIC was solved at 3.3 A in a closed-pore conformation, and GLIC at up to 2.9 A in an apparently open-pore conformation. These data reveal many structural features, notably the architecture of the pore, including its gate and its selectivity filter, and the interactions between the protein and lipids. In addition, comparison of the structures of GLIC and ELIC hints at a mechanism of channel opening, which consists of both a quaternary twist and a tertiary deformation. This mechanism couples opening-closing motions of the channel with a global reorganization of the protein, including the subunit interface that holds the neurotransmitter binding sites in eukaryotic pLGICs.
Assuntos
Proteínas de Bactérias/química , Ativação do Canal Iônico , Canais Iônicos/química , Receptores de Neurotransmissores/química , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Cristalografia por Raios X , Canais Iônicos/fisiologia , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptores de Neurotransmissores/fisiologiaRESUMO
Inaccurately perceived as niche drugs, antiemetics are key elements of cancer treatment alleviating the most dreaded side effect of chemotherapy. Serotonin 5-HT3 receptor antagonists are the most commonly prescribed class of drugs to control chemotherapy-induced nausea and vomiting. These antagonists have been clinically successful drugs since the 1980s, yet our understanding of how they operate at the molecular level has been hampered by the difficulty of obtaining structures of drug-receptor complexes. Here, we report the cryoelectron microscopy structure of the palonosetron-bound 5-HT3 receptor. We investigate the binding of palonosetron, granisetron, dolasetron, ondansetron, and cilansetron using molecular dynamics, covering the whole set of antagonists used in clinical practice. The structural and computational results yield detailed atomic insight into the binding modes of the drugs. In light of our data, we establish a comprehensive framework underlying the inhibition mechanism by the -setron drug family.