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The peptidoglycan (PG) cell wall protects bacteria against osmotic lysis and determines cell shape, making this structure a key antibiotic target. Peptidoglycan is a polymer of glycan chains connected by peptide crosslinks, and its synthesis requires precise spatiotemporal coordination between glycan polymerization and crosslinking. However, the molecular mechanism by which these reactions are initiated and coupled is unclear. Here we use single-molecule FRET and cryo-EM to show that an essential PG synthase (RodA-PBP2) responsible for bacterial elongation undergoes dynamic exchange between closed and open states. Structural opening couples the activation of polymerization and crosslinking and is essential in vivo. Given the high conservation of this family of synthases, the opening motion that we uncovered likely represents a conserved regulatory mechanism that controls the activation of PG synthesis during other cellular processes, including cell division. Coordination of cell wall assembly is critical for bacterial morphology and survival. Here, the authors show that activation of cell wall synthesis by the Rod complex is regulated by the structural dynamics of RodA-PBP2.

The ability to design functional sequences and predict effects of variation is central to protein engineering and biotherapeutics. State-of-art computational methods rely on models that leverage evolutionary information but are inadequate for important applications where multiple sequence alignments are not robust. Such applications include the prediction of variant effects of indels, disordered proteins, and the design of proteins such as antibodies due to the highly variable complementarity determining regions. We introduce a deep generative model adapted from natural language processing for prediction and design of diverse functional sequences without the need for alignments. The model performs state-of-art prediction of missense and indel effects and we successfully design and test a diverse 10 5 -nanobody library that shows better expression than a 1000-fold larger synthetic library. Our results demonstrate the power of the alignment-free autoregressive model in generalizing to regions of sequence space traditionally considered beyond the reach of prediction and design. The ability to design functional sequences is central to protein engineering and biotherapeutics. Here the authors introduce a deep generative alignment-free model for sequence design applied to highly variable regions and design and test a diverse nanobody library with improved properties for selection experiments.

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.

One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5-15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria.

The σ1 receptor is a poorly understood membrane protein expressed throughout the human body. Ligands targeting the σ1 receptor are in clinical trials for treatment of Alzheimer’s disease, ischemic stroke, and neuropathic pain. However, relatively little is known regarding the σ1 receptor’s molecular function. Here, we present crystal structures of human σ1 receptor bound to the antagonists haloperidol and NE-100, and the agonist (+)-pentazocine, at crystallographic resolutions of 3.1 Å, 2.9 Å, and 3.1 Å, respectively. These structures reveal a unique binding pose for the agonist. The structures and accompanying molecular dynamics (MD) simulations identify agonist-induced structural rearrangements in the receptor. Additionally, we show that ligand binding to σ1 is a multistep process that is rate limited by receptor conformational change. We used MD simulations to reconstruct a ligand binding pathway involving two major conformational changes. These data provide a framework for understanding the molecular basis for σ1 agonism.

Camelid single-domain antibody fragments (‘nanobodies’) provide the remarkable specificity of antibodies within a single 15-kDa immunoglobulin VHH domain. This unique feature has enabled applications ranging from use as biochemical tools to therapeutic agents. Nanobodies have emerged as especially useful tools in protein structural biology, facilitating studies of conformationally dynamic proteins such as G-protein-coupled receptors (GPCRs). Nearly all nanobodies available to date have been obtained by animal immunization, a bottleneck restricting many applications of this technology. To solve this problem, we report a fully in vitro platform for nanobody discovery based on yeast surface display. We provide a blueprint for identifying nanobodies, demonstrate the utility of the library by crystallizing a nanobody with its antigen, and most importantly, we utilize the platform to discover conformationally selective nanobodies to two distinct human GPCRs. To facilitate broad deployment of this platform, the library and associated protocols are freely available for nonprofit research.

Gram-negative bacteria are surrounded by an inner cytoplasmic membrane and by an outer membrane, which serves as a protective barrier to limit entry of many antibiotics. The distinctive properties of the outer membrane are due to the presence of lipopolysaccharide 1 . This large glycolipid, which contains numerous sugars, is made in the cytoplasm; a complex of proteins forms a membrane-to-membrane bridge that mediates transport of lipopolysaccharide from the inner membrane to the cell surface 1 . The inner-membrane components of the protein bridge comprise an ATP-binding cassette transporter that powers transport, but how this transporter ensures unidirectional lipopolysaccharide movement across the bridge to the outer membrane is unknown 2 . Here we describe two crystal structures of a five-component inner-membrane complex that contains all the proteins required to extract lipopolysaccharide from the membrane and pass it to the protein bridge. Analysis of these structures, combined with biochemical and genetic experiments, identifies the path of lipopolysaccharide entry into the cavity of the transporter and up to the bridge. We also identify a protein gate that must open to allow movement of substrate from the cavity onto the bridge. Lipopolysaccharide entry into the cavity is ATP-independent, but ATP is required for lipopolysaccharide movement past the gate and onto the bridge. Our findings explain how the inner-membrane transport complex controls efficient unidirectional transport of lipopolysaccharide against its concentration gradient. Crystal structures of a five-protein complex comprising the inner-membrane components of the bacterial lipopolysaccharide transporter provide insight into the mechanism of extraction of lipopolysaccharide from the inner membrane and its transport to the outer membrane.

Many approved drugs bind to G protein–coupled receptors (GPCRs). A challenge in targeting GPCRs is that different ligands preferentially activate different signaling pathways. Two papers show how biased signaling arises for the angiotensin II type 1 receptor that couples to two signaling partners (G proteins and arrestins). Suomivuori et al. used large-scale atomistic simulations to show that coupling to the two pathways is through two distinct GPCR conformations and that extracellular ligands favor one or the other conformation. Wingler et al. present crystal structures of the same receptor bound to ligands with different bias profiles. These structures show conformational changes in and around the binding pocket that match those observed in simulations. This work could provide a framework for the rational design of drugs that are more effective and have fewer side effects. Science , this issue p. 881 , p. 888 Atomic-level molecular dynamics reveals how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. Biased signaling, in which different ligands that bind to the same G protein–coupled receptor preferentially trigger distinct signaling pathways, holds great promise for the design of safer and more effective drugs. Its structural mechanism remains unclear, however, hampering efforts to design drugs with desired signaling profiles. Here, we use extensive atomic-level molecular dynamics simulations to determine how arrestin bias and G protein bias arise at the angiotensin II type 1 receptor. The receptor adopts two major signaling conformations, one of which couples almost exclusively to arrestin, whereas the other also couples effectively to a G protein. A long-range allosteric network allows ligands in the extracellular binding pocket to favor either of the two intracellular conformations. Guided by this computationally determined mechanism, we designed ligands with desired signaling profiles.

Many approved drugs bind to G protein–coupled receptors (GPCRs). A challenge in targeting GPCRs is that different ligands preferentially activate different signaling pathways. Two papers show how biased signaling arises for the angiotensin II type 1 receptor that couples to two signaling partners (G proteins and arrestins). Suomivuori et al. used large-scale atomistic simulations to show that coupling to the two pathways is through two distinct GPCR conformations and that extracellular ligands favor one or the other conformation. Wingler et al. present crystal structures of the same receptor bound to ligands with different bias profiles. These structures show conformational changes in and around the binding pocket that match those observed in simulations. This work could provide a framework for the rational design of drugs that are more effective and have fewer side effects. Science , this issue p. 881 , p. 888 Crystal structures elucidate how ligands select among signaling conformations of a G protein–coupled receptor. Biased agonists of G protein–coupled receptors (GPCRs) preferentially activate a subset of downstream signaling pathways. In this work, we present crystal structures of angiotensin II type 1 receptor (AT1R) (2.7 to 2.9 angstroms) bound to three ligands with divergent bias profiles: the balanced endogenous agonist angiotensin II (AngII) and two strongly β-arrestin–biased analogs. Compared with other ligands, AngII promotes more-substantial rearrangements not only at the bottom of the ligand-binding pocket but also in a key polar network in the receptor core, which forms a sodium-binding site in most GPCRs. Divergences from the family consensus in this region, which appears to act as a biased signaling switch, may predispose the AT1R and certain other GPCRs (such as chemokine receptors) to adopt conformations that are capable of activating β-arrestin but not heterotrimeric G q protein signaling.

The σ 2 receptor has attracted intense interest in cancer imaging 1 , psychiatric disease 2 , neuropathic pain 3 – 5 and other areas of biology 6 , 7 . Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone 2 and the tool compound PB28 8 . These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 μM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ 1 receptor. Crystal structures of two ligands bound to the σ 2 receptor confirmed the docked poses. To investigate the contribution of the σ 2 receptor in pain, two potent σ 2 -selective ligands and one potent σ 1 /σ 2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model 9 , suggesting that the σ 2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology. Crystal structures of the σ 2 receptor are determined and used to perform a docking screen of nearly 500 million molecules, identifying σ 2 -selective ligands and providing insight into the role of σ 2 in neuropathic pain.