Optimization of membrane protein stability under different solution conditions is essential for obtaining crystals that diffract to high resolution. Traditional methods that evaluate protein ...stability require large amounts of material and are, therefore, ill suited for medium- to high-throughput screening of membrane proteins. Here we present a rapid and efficient fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS). In this method, the target protein is fused to GFP. Heated protein samples, treated with a panel of additives, are then analyzed by FSEC. FSEC-TS allows one to evaluate the thermostability of nanogram-to-microgram amounts of the target protein under a variety of conditions without purification. We applied this method to the Danio rerio P2X4 receptor and Caenorhabditis elegans GluCl to screen ligands, ions, and lipids, including newly designed cholesterol derivatives. In the case of GluCl, the screening results were used to obtain crystals of the receptor in the presence of lipids.
► Membrane protein precrystallization screening ► Fluorescence-detection size-exclusion chromatography-based thermostability assay
Structures of membrane proteins are of great interest but difficult to obtain. Now, Hattori et al. present a fluorescence-detection size-exclusion chromatography-based thermostability assay that allows for HTS on a small scale to identify conditions that stabilize protein for structure determination.
Fast inhibitory neurotransmission is essential for nervous system function and is mediated by binding of inhibitory neurotransmitters to receptors of the Cys-loop family embedded in the membranes of ...neurons. Neurotransmitter binding triggers a conformational change in the receptor, opening an intrinsic chloride channel and thereby dampening neuronal excitability. Here we present the first three-dimensional structure, to our knowledge, of an inhibitory anion-selective Cys-loop receptor, the homopentameric Caenorhabditis elegans glutamate-gated chloride channel α (GluCl), at 3.3 Å resolution. The X-ray structure of the GluCl-Fab complex was determined with the allosteric agonist ivermectin and in additional structures with the endogenous neurotransmitter L-glutamate and the open-channel blocker picrotoxin. Ivermectin, used to treat river blindness, binds in the transmembrane domain of the receptor and stabilizes an open-pore conformation. Glutamate binds in the classical agonist site at subunit interfaces, and picrotoxin directly occludes the pore near its cytosolic base. GluCl provides a framework for understanding mechanisms of fast inhibitory neurotransmission and allosteric modulation of Cys-loop receptors.
GABAA receptors are pentameric ligand-gated ion channels that mediate most fast neuronal inhibition in the brain. In addition to their important physiological roles, they are noteworthy in their rich ...pharmacology; prominent drugs used for anxiety, insomnia, and general anesthesia act through positive modulation of GABAA receptors. Direct structural information for how these drugs work was absent until recently. Efforts in structural biology over the past few years have revealed how important drug classes and natural products interact with the GABAA receptor, providing a foundation for studies in dynamics and structure-guided drug design. Here, we review recent developments in GABAA receptor structural pharmacology, focusing on subunit assemblies of the receptor found at synapses.
GABAA receptors are ion channels important in brain function and are the target of chemically diverse and clinically important drugs for insomnia, epilepsy, and anesthesia.Recent structures of GABAA receptors have revealed detailed interactions of the neurotransmitter GABA, benzodiazepines, general anesthetics, and antagonists.Intravenous anesthetics bind in both common and distinctive membrane sites.Electrophysiological and molecular dynamics studies build off the static structures, to probe mechanisms of potentiation and inhibition by chemically diverse compounds.
GABA
receptors are pentameric ligand-gated ion channels that mediate most fast neuronal inhibition in the brain. In addition to their important physiological roles, they are noteworthy in their rich ...pharmacology; prominent drugs used for anxiety, insomnia, and general anesthesia act through positive modulation of GABA
receptors. Direct structural information for how these drugs work was absent until recently. Efforts in structural biology over the past few years have revealed how important drug classes and natural products interact with the GABA
receptor, providing a foundation for studies in dynamics and structure-guided drug design. Here, we review recent developments in GABA
receptor structural pharmacology, focusing on subunit assemblies of the receptor found at synapses.
Nicotinic acetylcholine receptors are ligand-gated ion channels that mediate fast chemical neurotransmission at the neuromuscular junction and have diverse signalling roles in the central nervous ...system. The nicotinic receptor has been a model system for cell-surface receptors, and specifically for ligand-gated ion channels, for well over a century. In addition to the receptors' prominent roles in the development of the fields of pharmacology and neurobiology, nicotinic receptors are important therapeutic targets for neuromuscular disease, addiction, epilepsy and for neuromuscular blocking agents used during surgery. The overall architecture of the receptor was described in landmark studies of the nicotinic receptor isolated from the electric organ of Torpedo marmorata. Structures of a soluble ligand-binding domain have provided atomic-scale insights into receptor-ligand interactions, while high-resolution structures of other members of the pentameric receptor superfamily provide touchstones for an emerging allosteric gating mechanism. All available high-resolution structures are of homopentameric receptors. However, the vast majority of pentameric receptors (called Cys-loop receptors in eukaryotes) present physiologically are heteromeric. Here we present the X-ray crystallographic structure of the human α4β2 nicotinic receptor, the most abundant nicotinic subtype in the brain. This structure provides insights into the architectural principles governing ligand recognition, heteromer assembly, ion permeation and desensitization in this prototypical receptor class.
Cys-loop receptors are neurotransmitter-gated ion channels that are essential mediators of fast chemical neurotransmission and are associated with a large number of neurological diseases and ...disorders, as well as parasitic infections. Members of this ion channel superfamily mediate excitatory or inhibitory neurotransmission depending on their ligand and ion selectivity. Structural information for Cys-loop receptors comes from several sources including electron microscopic studies of the nicotinic acetylcholine receptor, high-resolution X-ray structures of extracellular domains and X-ray structures of bacterial orthologues. In 2011 our group published structures of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in complex with the allosteric partial agonist ivermectin, which provided insights into the structure of a possibly open state of a eukaryotic Cys-loop receptor, the basis for anion selectivity and channel block, and the mechanism by which ivermectin and related molecules stabilize the open state and potentiate neurotransmitter binding. However, there remain unanswered questions about the mechanism of channel opening and closing, the location and nature of the shut ion channel gate, the transitions between the closed/resting, open/activated and closed/desensitized states, and the mechanism by which conformational changes are coupled between the extracellular, orthosteric agonist binding domain and the transmembrane, ion channel domain. Here we present two conformationally distinct structures of C. elegans GluCl in the absence of ivermectin. Structural comparisons reveal a quaternary activation mechanism arising from rigid-body movements between the extracellular and transmembrane domains and a mechanism for modulation of the receptor by phospholipids.
The ability of oligomeric membrane proteins to assemble in different functional ratios of subunits is a common feature across many systems. Recombinant expression of hetero-oligomeric proteins with ...defined stoichiometries facilitates detailed structural and functional analyses, but remains a major challenge. Here we present two methods for overcoming this challenge: one for rapid virus titration and another for stoichiometry determination. When these methods are coupled, they allow for efficient dissection of the heteromer stoichiometry problem and optimization of homogeneous protein expression. We demonstrate the utility of the methods in a system that to date has proved resistant to atomic-scale structural study, the nicotinic acetylcholine receptor. Leveraging these two methods, we have successfully expressed, purified, and grown diffraction-quality crystals of this challenging target.
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•Streamlined bacmam virus titration method•Fluorescent protein fusion approach for estimation of subunit ratio in a heteromer•Expression and crystallization of a defined nicotinic receptor stoichiometry
Morales-Perez et al. developed a pair of methods to efficiently express defined stoichiometries of heteromeric membrane proteins. Application of this approach to a nicotinic acetylcholine receptor that can assemble in multiple functional ratios of subunits has yielded diffraction-quality crystals of the receptor.
Fast inhibitory neurotransmission in the brain is principally mediated by the neurotransmitter GABA (γ-aminobutyric acid) and its synaptic target, the type A GABA receptor (GABA
receptor). ...Dysfunction of this receptor results in neurological disorders and mental illnesses including epilepsy, anxiety and insomnia. The GABA
receptor is also a prolific target for therapeutic, illicit and recreational drugs, including benzodiazepines, barbiturates, anaesthetics and ethanol. Here we present high-resolution cryo-electron microscopy structures of the human α1β2γ2 GABA
receptor, the predominant isoform in the adult brain, in complex with GABA and the benzodiazepine site antagonist flumazenil, the first-line clinical treatment for benzodiazepine overdose. The receptor architecture reveals unique heteromeric interactions for this important class of inhibitory neurotransmitter receptor. This work provides a template for understanding receptor modulation by GABA and benzodiazepines, and will assist rational approaches to therapeutic targeting of this receptor for neurological disorders and mental illness.
Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABA
) receptors to dampen neuronal activity in the brain
. However, direct ...structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABA
receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABA
receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABA
receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.
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