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Type-A γ-aminobutyric acid receptors (GABA A Rs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABA A R signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABA A R, the human β3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABA A R modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission. GABA A receptors are the principal mediators of rapid inhibitor synaptic transmission in the brain, and a decline in GABA A signalling leads to diseases including epilepsy, insomnia, anxiety and autism; here, the first X-ray crystal structure of a human GABA A receptor, the human β3 homopentamer, reveals structural features unique for this receptor class and uncovers the locations of key disease-causing mutations. GABA A receptor structure Paul Miller and Radu Aricescu report the first X-ray crystal structure of the human GABA A receptor, a pentameric ligand-gated ion channel and the principal mediator of rapid inhibitory synaptic transmission in the brain. The overall structure resembles those of other Cys-loop receptors but there are also several unique features, including the presence of an extended glycan sheath that would restrict interactions with other synaptic proteins. The authors discuss how specific mutations may be linked to specific diseases, and since the structure was obtained in the presence of benzamidine, a GABA A receptor agonist, it is hoped that this work could contribute to the design of new therapeutic agents.

Type A γ-aminobutyric acid (GABA A ) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system 1 , 2 . Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia 3 , 4 . Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1β2/3γ2 GABA A receptors 5 . The β3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in β1–β3 triple knockout neurons 6 . So far, efforts to generate accurate structural models for heteromeric GABA A receptors have been hampered by the use of engineered receptors and the presence of detergents 7 – 9 . Notably, some recent cryo-electron microscopy reconstructions have reported ‘collapsed’ conformations 8 , 9 ; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor 10 , 11 , the large body of structural work on homologous homopentameric receptor variants 12 and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1β3γ2L—a major synaptic GABA A receptor isoform—that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator ‘megabody’ and is in a desensitized conformation. Each GABA A receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3–M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABA A receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology. A high-resolution cryo-electron microscopy structure is reported for the full-length human α1β3γ2L GABA A receptor, functionally reconstituted in lipid nanodiscs.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3–4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

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.Megabodies, built by grafting nanobodies onto larger protein scaffolds, help alleviate problems of particle size and preferential orientation at the water–air interfaces during cryo-EM based structure determination experiments and are shown to be generalizable to soluble and membrane-bound proteins.

Crystal structures and functional assays of a chimeric GABA A receptor in apo and pregnanolone-bound states reveal how neurosteroid binding alters receptor conformation to modulate channel opening. Type A γ-aminobutyric acid receptors (GABA A Rs) are the principal mediators of inhibitory neurotransmission in the human brain. Endogenous neurosteroids interact with GABA A Rs to regulate acute and chronic anxiety and are potent sedative, analgesic, anticonvulsant and anesthetic agents. Their mode of binding and mechanism of receptor potentiation, however, remain unknown. Here we report crystal structures of a chimeric GABA A R construct in apo and pregnanolone-bound states. The neurosteroid-binding site is mechanically coupled to the helices lining the ion channel pore and modulates the desensitization-gate conformation. We demonstrate that the equivalent site is responsible for physiological, heteromeric GABA A R potentiation and explain the contrasting modulatory properties of 3a versus 3b neurosteroid epimers. These results illustrate how peripheral lipid ligands can regulate the desensitization gate of GABA A Rs, a process of broad relevance to pentameric ligand-gated ion channels.

Ionotropic glutamate receptor (iGIuR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGIuR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGIuR δ2 (GIuD2) and presynaptic β-neurexin 1 (β-NRX1) via CbIn1, a C1q-like synaptic organizer. We show how CbIn1 hexamers \"anchor\" GIuD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D-serine–dependent GIuD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber–Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGIuR function.

Analysis of proteins within their native environment can confirm and extend in vitro –derived conclusions. NMR analysis of superoxide dismutase 1 in live human cells now corroborates previously identified steps on the maturation pathway and demonstrates copper-independent function of the chaperone CCS. We use NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 oxidizes the SOD1 intrasubunit disulfide bond through both copper-dependent and copper-independent mechanisms. Our approach represents a new strategy for structural investigation of endogenously expressed proteins in a physiological (cellular) environment.

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers. Human cells have two copies of each chromosome: one from the mother, and one from the father. When cells divide to form sex cells, such as sperm or egg cells, the maternal and paternal chromosomes line up next to each other and swap some of their DNA. This process, known as genetic recombination, creates different versions of genes and ensures that we are all unique – or genetically diverse. Recombination is a complex process that is largely controlled by a protein called PRDM9. This protein binds DNA at particular spots on the chromosome and directs other proteins to carry out recombination nearby. However, not all of PRDM9’s binding sites are known, and not all regions that PRDM9 binds to undergo recombination. Until now, it was not understood why this is the case at fine scales. To investigate this further, Altemose et al. activated the human version of PRDM9 in human kidney cells grown in the laboratory. The results showed that PRDM9 often bound near the start sites of genes, although these regions rarely undergo recombination in humans. When PRDM9 bound near these sites, it sometimes turned the gene on, which suggests that it may also help to regulate the activity of genes. Moreover, a specific group of DNA-binding proteins, called KRAB-ZNF proteins, appear to suppress recombination wherever they bind, which explains why some PRDM9 binding sites do not recombine. Lastly, Altemose et al. discovered that the part of PRDM9 that binds to DNA can also bind to other copies of PRDM9 proteins. This self-binding ability might play a role in bringing together the maternal and paternal chromosomes at the correct spots during recombination. Together, these results shed new light on the recombination process, which is a driving force in the formation of new species and essential for fertility. A next step will be to study these results further in tissues of the reproductive organs. This will provide a better understanding of the forces that shape human evolution.

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor.

Eph receptors are cell surface protein tyrosine kinases that mediate cell-cell communication. The crystal structure of the full ectodomain of unliganded human EphA2 (eEphA2) reveals that it forms linear arrays of staggered, parallel receptors, whereas that of eEphA2 in complex with ephrinA5 forms a more elaborate assembly with interfaces that are crucial for localization at cell-cell contacts and for activation-dependent degradation. Erythropoetin-producing hepatoma (Eph) receptors are cell-surface protein tyrosine kinases mediating cell-cell communication. Upon activation, they form signaling clusters. We report crystal structures of the full ectodomain of human EphA2 (eEphA2) both alone and in complex with the receptor-binding domain of the ligand ephrinA5 (ephrinA5 RBD). Unliganded eEphA2 forms linear arrays of staggered parallel receptors involving two patches of residues conserved across A-class Ephs. eEphA2–ephrinA5 RBD forms a more elaborate assembly, whose interfaces include the same conserved regions on eEphA2, but rearranged to accommodate ephrinA5 RBD. Cell-surface expression of mutant EphA2s showed that these interfaces are critical for localization at cell-cell contacts and activation-dependent degradation. Our results suggest a 'nucleation' mechanism whereby a limited number of ligand-receptor interactions 'seed' an arrangement of receptors which can propagate into extended signaling arrays.