Explore the vast range of titles available.

N -methyl- d -aspartate receptors (NMDARs) are heterotetrameric ion channels that initiate chemical and electrical signals in postsynaptic cells. They play key roles in brain development and function and are the targets of drugs for treating neurological disorders such as schizophrenia, depression, and epilepsy. For the channel to open, it must bind glutamate and glycine and release a blocking magnesium ion. Most NMDARs have three different subunits that bind glycine and glutamine, but structural studies have focused on tetramers of only two subunits. Lü et al. determined the structure of triheteromeric NMDAR. The structural studies show how having three different subunits modifies receptor symmetry and subunit interactions and increases the complexity of receptor regulation. Science , this issue p. eaal3729 Having three different subunits allows complex regulation of the neuronal NMDA ionotropic glutamate receptor involved in synaptic plasticity. N -methyl- d -aspartate receptors (NMDARs) are heterotetrameric ion channels assembled as diheteromeric or triheteromeric complexes. Here, we report structures of the triheteromeric GluN1/GluN2A/GluN2B receptor in the absence or presence of the GluN2B-specific allosteric modulator Ro 25-6981 (Ro), determined by cryogenic electron microscopy (cryo-EM). In the absence of Ro, the GluN2A and GluN2B amino-terminal domains (ATDs) adopt “closed” and “open” clefts, respectively. Upon binding Ro, the GluN2B ATD clamshell transitions from an open to a closed conformation. Consistent with a predominance of the GluN2A subunit in ion channel gating, the GluN2A subunit interacts more extensively with GluN1 subunits throughout the receptor, in comparison with the GluN2B subunit. Differences in the conformation of the pseudo-2-fold–related GluN1 subunits further reflect receptor asymmetry. The triheteromeric NMDAR structures provide the first view of the most common NMDA receptor assembly and show how incorporation of two different GluN2 subunits modifies receptor symmetry and subunit interactions, allowing each subunit to uniquely influence receptor structure and function, thus increasing receptor complexity.

The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel 1 . Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans . The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid–protein interactions in the mechanism by which mechanical force is transduced to ion channel gating. Structural studies of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction channel complex of Caenorhabditis elegans reveal the subunit composition and the roles of protein–membrane interactions in the conversion of mechanical force to ion channel activity.

It can be difficult to express large amounts of membrane proteins for structural analysis. Using pEG BacMam it is possible to screen potential candidate proteins as well as to scale up expression in mammalian cells. Structural, biochemical and biophysical studies of eukaryotic membrane proteins are often hampered by difficulties in overexpression of the candidate molecule. Baculovirus transduction of mammalian cells (BacMam), although a powerful method to heterologously express membrane proteins, can be cumbersome for screening and expression of multiple constructs. We therefore developed plasmid Eric Gouaux (pEG) BacMam, a vector optimized for use in screening assays, as well as for efficient production of baculovirus and robust expression of the target protein. In this protocol, we show how to use small-scale transient transfection and fluorescence-detection size-exclusion chromatography (FSEC) experiments using a GFP-His 8 –tagged candidate protein to screen for monodispersity and expression level. Once promising candidates are identified, we describe how to generate baculovirus, transduce HEK293S GnTI − ( N -acetylglucosaminyltransferase I-negative) cells in suspension culture and overexpress the candidate protein. We have used these methods to prepare pure samples of chicken acid-sensing ion channel 1a (cASIC1) and Caenorhabditis elegans glutamate-gated chloride channel (GluCl) for X-ray crystallography, demonstrating how to rapidly and efficiently screen hundreds of constructs and accomplish large-scale expression in 4–6 weeks.

Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.

Hearing and balance involve the transduction of mechanical stimuli into electrical signals by deflection of bundles of stereocilia linked together by protocadherin 15 (PCDH15) and cadherin 23 ‘tip links’. PCDH15 transduces tip link tension into opening of a mechano-electrical transduction (MET) ion channel. PCDH15 also interacts with LHFPL5, a candidate subunit of the MET channel. Here we illuminate the PCDH15-LHFPL5 structure, showing how the complex is composed of PCDH15 and LHFPL5 subunit pairs related by a 2-fold axis. The extracellular cadherin domains define a mobile tether coupled to a rigid, 2-fold symmetric ‘collar’ proximal to the membrane bilayer. LHFPL5 forms extensive interactions with the PCDH15 transmembrane helices and stabilizes the overall PCDH15-LHFPL5 assembly. Our studies illuminate the architecture of the PCDH15-LHFPL5 complex, localize mutations associated with deafness, and shed new light on how forces in the PCDH15 tether may be transduced into the stereocilia membrane.

Mechanosensory transduction (MT), the conversion of mechanical stimuli into electrical signals, underpins hearing and balance and is carried out within hair cells in the inner ear. Hair cells harbor actin-filled stereocilia, arranged in rows of descending heights, where the tips of stereocilia are connected to their taller neighbors by a filament composed of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), deemed the ‘tip link.’ Tension exerted on the tip link opens an ion channel at the tip of the shorter stereocilia, thus converting mechanical force into an electrical signal. While biochemical and structural studies have provided insights into the molecular composition and structure of isolated portions of the tip link, the architecture, location, and conformational states of intact tip links, on stereocilia, remains unknown. Here, we report in situ cryo-electron microscopy imaging of the tip link in mouse stereocilia. We observe individual PCDH15 molecules at the tip and shaft of stereocilia and determine their stoichiometry, conformational heterogeneity, and their complexes with other filamentous proteins, perhaps including CDH23. The PCDH15 complexes occur in clusters, frequently with more than one copy of PCDH15 at the tip of stereocilia, suggesting that tip links might consist of more than one copy of PCDH15 complexes and, by extension, might include multiple MT complexes.

Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans . We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and ‘Protein S’), from transgenic C. elegans , demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes. Key points This protocol outlines the isolation of membrane protein complexes from transgenic C. elegans The primary advantage of the protocol is that it enables isolation of sufficient quantities of low-abundance native membrane protein complexes for use in structural, biochemical or biophysical studies The authors provide a detailed protocol for the isolation of four membrane protein complexes (transmembrane channel-like proteins 1 and 2, lipid transfer protein and ‘Protein S’) from transgenic C. elegans .

N -methyl- d -aspartate (NMDA) receptors are Hebbian-like coincidence detectors, requiring binding of glycine and glutamate in combination with the relief of voltage-dependent magnesium block to open an ion conductive pore across the membrane bilayer. Despite the importance of the NMDA receptor in the development and function of the brain, a molecular structure of an intact receptor has remained elusive. Here we present X-ray crystal structures of the Xenopus laevis GluN1–GluN2B NMDA receptor with the allosteric inhibitor, Ro25-6981, partial agonists and the ion channel blocker, MK-801. Receptor subunits are arranged in a 1-2-1-2 fashion, demonstrating extensive interactions between the amino-terminal and ligand-binding domains. The transmembrane domains harbour a closed-blocked ion channel, a pyramidal central vestibule lined by residues implicated in binding ion channel blockers and magnesium, and a ∼twofold symmetric arrangement of ion channel pore loops. These structures provide new insights into the architecture, allosteric coupling and ion channel function of NMDA receptors. X-ray crystal structures are presented of the N -methyl- d -aspartate (NMDA) receptor, a calcium-permeable ion channel that opens upon binding of glutamate and glycine; glutamate is a key excitatory neurotransmitter and enhanced structural insight of this receptor may aid development of therapeutic small molecules. NMDA receptor pore architecture Glutamate is the primary excitatory neurotransmitter in the central nervous system, acting at ionotropic and metabotropic glutamate receptors. Ionotropic glutamate receptors function by opening a transmembrane ion channel upon binding of glutamate. Eric Gouaux and colleagues report the X-ray crystal structure of the GluN1–GluN2B N -methyl- D -aspartate (NMDA) receptor, a heterotetrameric complex comprised of a glycine-binding GluN1 subunit and a glutamate-binding GluN2 subunit. Activation of the receptor opens a cation-selective, calcium-permeable channel, thereby causing further depolarization of the cell membrane and influx of calcium. The structure of this membrane protein was obtained in complex with the allosteric inhibitor Ro25-6981, the channel blocker MK-801, and with two partial agonists. The overall structure of this membrane protein is mushroom-like, with the GluN1 and GluN2B subunits arranged as a dimer-of-dimers in a 1-2-1-2 fashion.

LeuT, a bacterial homologue of eukaryotic biogenic amine transporters (BATs), is engineered to harbour human BAT-like pharmacology by the mutation of key residues around the primary binding pocket; this mutant is able to bind several classes of antidepressant drug with high affinity, helping to define their common mechanisms of action. Structural approach to antidepressant activity Neurotransmitter sodium symporters (NSSs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents, including selective serotonin reuptake inhibitors (SSRIs), serotonin–noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). An X-ray crystal structure of a eukaryotic NSS is not available, hindering our understanding of the mechanism of action of these antidepressants. In this manuscript, the authors used a bacterial homologue of NSSs as a scaffold to generate a hybrid protein with a pharmacological profile very similar to that of biogenic amine transporters. They solved X-ray crystal structures of these 'LeuBAT' variants in the presence of four SSRIs, two SNRIs, a TCA and the stimulant mazindol. Even though these compounds have very different chemical structures, they all bind at the same site of LeuBAT, thereby enabling the authors to better understand how SSRIs, SNRIs and TCAs bind to their eukaryotic NSS targets. The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin–noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs) 1 , 2 . Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function 3 . However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter 4 , it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT 5 , 6 , 7 and act as non-competitive inhibitors of transport 5 . By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 . Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.