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The human σ 1 receptor is an enigmatic endoplasmic-reticulum-resident transmembrane protein implicated in a variety of disorders including depression, drug addiction, and neuropathic pain 1 . Recently, an additional connection to amyotrophic lateral sclerosis has emerged from studies of human genetics and mouse models 2 . Unlike many transmembrane receptors that belong to large, extensively studied families such as G-protein-coupled receptors or ligand-gated ion channels, the σ 1 receptor is an evolutionary isolate with no discernible similarity to any other human protein. Despite its increasingly clear importance in human physiology and disease, the molecular architecture of the σ 1 receptor and its regulation by drug-like compounds remain poorly defined. Here we report crystal structures of the human σ 1 receptor in complex with two chemically divergent ligands, PD144418 and 4-IBP. The structures reveal a trimeric architecture with a single transmembrane domain in each protomer. The carboxy-terminal domain of the receptor shows an extensive flat, hydrophobic membrane-proximal surface, suggesting an intimate association with the cytosolic surface of the endoplasmic reticulum membrane in cells. This domain includes a cupin-like β-barrel with the ligand-binding site buried at its centre. This large, hydrophobic ligand-binding cavity shows remarkable plasticity in ligand recognition, binding the two ligands in similar positions despite dissimilar chemical structures. Taken together, these results reveal the overall architecture, oligomerization state, and molecular basis for ligand recognition by this important but poorly understood protein. The X-ray crystal structures of the human σ1 receptor bound to two different ligands are reported, revealing the overall architecture, oligomerization state, and molecular basis for ligand recognition by this protein. Structure of human σ1 receptor The human σ1 receptor is a transmembrane protein of the endoplasmic reticulum that is implicated in depression, drug addiction and neurodegenerative disorders including amyotrophic lateral sclerosis. In this manuscript, the authors report X-ray crystal structures of the human σ1 receptor in the presence of two different ligands. The structure of the protein is trimeric, with a single transmembrane domain in each protomer. Despite having dissimilar chemical structures, both ligands bind to the same site, a large, hydrophobic cavity. Taken together, these results reveal the overall architecture, oligomerization state, and molecular basis for ligand recognition by this important, but poorly understood, protein.

Background Genetic variation in the human genome is a major determinant of individual disease risk, but the vast majority of missense variants have unknown etiological effects. Here, we present a robust learning framework for leveraging saturation mutagenesis experiments to construct accurate computational predictors of proteome-wide missense variant pathogenicity. Results We train cross-protein transfer (CPT) models using deep mutational scanning (DMS) data from only five proteins and achieve state-of-the-art performance on clinical variant interpretation for unseen proteins across the human proteome. We also improve predictive accuracy on DMS data from held-out proteins. High sensitivity is crucial for clinical applications and our model CPT-1 particularly excels in this regime. For instance, at 95% sensitivity of detecting human disease variants annotated in ClinVar, CPT-1 improves specificity to 68%, from 27% for ESM-1v and 55% for EVE. Furthermore, for genes not used to train REVEL, a supervised method widely used by clinicians, we show that CPT-1 compares favorably with REVEL. Our framework combines predictive features derived from general protein sequence models, vertebrate sequence alignments, and AlphaFold structures, and it is adaptable to the future inclusion of other sources of information. We find that vertebrate alignments, albeit rather shallow with only 100 genomes, provide a strong signal for variant pathogenicity prediction that is complementary to recent deep learning-based models trained on massive amounts of protein sequence data. We release predictions for all possible missense variants in 90% of human genes. Conclusions Our results demonstrate the utility of mutational scanning data for learning properties of variants that transfer to unseen proteins.

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes. The determination of high resolution structures of G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins is challenging. Here authors develop an antibody fragment, mAB16, which stabilizes GPCR/G-protein complexes and facilitates the application of high resolution cryo-EM.

The μ-opioid receptor (μOR), a prototypical G protein-coupled receptor (GPCR), is the target of opioid analgesics such as morphine and fentanyl. Due to the severe side effects of current opioid drugs, there is considerable interest in developing novel modulators of μOR function. Most GPCR ligands today are small molecules, however biologics, including antibodies and nanobodies, represent alternative therapeutics with clear advantages such as affinity and target selectivity. Here, we describe the nanobody NbE, which selectively binds to the μOR and acts as an antagonist. We functionally characterize NbE as an extracellular and genetically encoded μOR ligand and uncover the molecular basis for μOR antagonism by determining the cryo-EM structure of the NbE-μOR complex. NbE displays a unique ligand binding mode and achieves μOR selectivity by interactions with the orthosteric pocket and extracellular receptor loops. Based on a β-hairpin loop formed by NbE that deeply protrudes into the μOR, we design linear and cyclic peptide analogs that recapitulate NbE’s antagonism. The work illustrates the potential of nanobodies to uniquely engage with GPCRs and describes lower molecular weight μOR ligands that can serve as a basis for therapeutic developments. The µ-opioid receptor is a key clinical target. Here, the authors describe nanobody NbE, a selective and high affinity antagonist, which is downsized to small cyclic peptides. The work enables unique receptor targeting based on nanobody interaction.

Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl– for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change – which occurs in all conventional transporter mechanisms – has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl– substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl– transport that reconciles existing data on all CLC-type proteins. Cells are shielded from harmful molecules and other threats by a thin, flexible layer called the membrane. However, this barrier also prevents chloride, sodium, protons and other ions from moving in or out of the cell. Channels and transporters are two types of membrane proteins that form passageways for these charged particles. Channels let ions flow freely from one side of the membrane to the other. To do so, these proteins change their three-dimensional shape to open or close as needed. On the other hand, transporters actively pump ions across the membrane to allow the charged particles to accumulate on one side. The shape changes needed for that type of movement are different: the transporters have to open a passageway on one side of the membrane while closing it on the other side, alternating openings to one side or the other. In general, channels and transporters are not related to each other, but one exception is a group called CLCs proteins. Present in many organisms, this family contains a mixture of channels and transporters. For example, humans have nine CLC proteins: four are channels that allow chloride ions in and out, and five are ‘exchange transporters’ that make protons and chloride ions cross the membrane in opposite directions. These proteins let one type of charged particle move freely across the membrane, which generates energy that the transporter then uses to actively pump the other ion in the direction needed by the cell. Yet, the exact three-dimensional changes required for CLC transporters and channels to perform their roles are still unknown. To investigate this question, Chavan, Cheng et al. harnessed a technique called X-ray crystallography, which allows scientists to look at biological molecules at the level of the atom. This was paired with other methods to examine a CLC mutant that adopts the shape of a normal CLC transporter when it is loaded with a proton. The experiments revealed how various elements in the transporter move relative to each other to adopt a structure that allows protons and chloride ions to enter the protein from opposite sides of the membrane, using separate pathways. While obtained on a bacterial CLC, these results can be applied to other CLC channels and transporters (including those in humans), shedding light on how this family transports charged particles across membranes. From bone diseases to certain types of seizures, many human conditions are associated with poorly functioning CLCs. Understanding the way these structures change their shapes to perform their roles could help to design new therapies for these health problems.

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein G i . Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free G i . DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR–G i complex to previously determined structures of other GPCRs bound to the stimulatory G protein G s reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the G i protein-coupling specificity of the µOR. A cryo-electron structure of the µ-opioid receptor in complex with the peptide agonist DAMGO and the inhibitory G protein G i reveals structural determinants of its G protein-binding specificity.

Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains—the 7-transmembrane domains—in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling. The activation mechanism of metabotropic glutamate receptor subtype 5, a member of the family C G-protein-coupled receptors, is characterized by a combination of cryo-electron microscopy, crystallography and signalling studies.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain 1 . Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain 2 . In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding. We propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5, including a series of structures in lipid nanodiscs, from inactive to fully active, with agonist-bound intermediate states.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain (ECD) that is linked via a cysteine-rich domain (CRDs) to their 7-transmembrane (TM) domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the ECD to the G protein-coupling TM. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in nanodisc, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging we reveal distinct receptor conformations upon allosteric modulator and G protein binding.