Explore the vast range of titles available.

The enzyme inositol phosphorylceramide (IPC) synthase is essential for survival and virulence in fungi, while absent in mammals, thus representing a potential target for antifungal treatments. Aureobasidin A (AbA), a natural cyclic peptide, displays antifungal activity and inhibits IPC synthase, but the precise molecular mechanism remains unclear. Here, we present the cryo-EM structure of the Saccharomyces cerevisiae IPC synthase, composed of catalytic subunit Aur1 and regulatory subunit Kei1, in its AbA-bound state. The complex is resolved as a dimer of Aur1-Kei1 heterodimers, with Aur1 mediating homodimerization. AbA occupies a predominantly hydrophobic pocket in the catalytic core domain of each Aur1 subunit, blocking the entry of both substrates. Mutations conferring AbA resistance cluster near the AbA-binding site, thus interfering with AbA binding. Our study lays a foundation for the development of therapeutic drugs targeting fungal IPC synthase. The antifungal peptide aureobasidin A (AbA) inhibits a fungal enzyme, IPC synthase, through unclear mechanisms. Here, the authors present the cryo-EM structure of the yeast IPC synthase bound to AbA, revealing interactions between catalytic and regulatory subunits and showing how mutations conferring AbA resistance interfere with AbA binding.

A CRISPR screening approach shows that endoplasmic reticulum (ER)-associated protein complexes, including the oligosaccharyltransferase (OST) protein complex, are important for infection by dengue virus and other related mosquito-borne flaviviruses, whereas hepatitis C virus is dependent on distinct entry factors, RNA binding proteins and FAD biosynthesis. Host factors required for flavivirus infection Jan Carette and colleagues use a CRISPR screening approach to identify cellular genes with important roles in the lifecycle of two important human flaviviruses: dengue virus and hepatitis C virus. The authors show that endoplasmic-reticulum-associated protein complexes, including the oligosaccharyltransferase (OST) protein complex, are important for infection by dengue virus and other related mosquito-borne flaviviruses, whereas hepatitis C virus is dependent on distinct entry factors, RNA binding proteins and FAD biosynthesis. Also in this issue of Nature , Michael Diamond and colleagues report that the endoplasmic-reticulum-associated signal peptidase complex is required for infection by numerous flaviviruses, including West Nile, dengue and Zika viruses, but not for infection by other types of virus or for host protein synthesis. The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide 1 . No approved antivirals are available to date, and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates 2 . Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available 3 . Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies 4 . A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy 5 , 6 to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N -linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects 7 . By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-dependency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

Fungal inositol phosphorylceramide (IPC) synthase is an essential enzyme complex that catalyzes a critical step in sphingolipid biosynthesis. It is the molecular target of potent antifungal aureobasidin A (AbA). Despite its therapeutic relevance, the lack of structural and mechanistic insights into IPC synthase function and inhibition has impeded rational antifungal drug development. Here, we present cryo-EM structures of Saccharomyces cerevisiae IPC synthase in two distinct functional states: a ceramide-bound form and an AbA-inhibited complex. Our study reveals a conserved heterodimeric architecture formed by Aur1 and Kei1, stabilized through extensive protein-protein and lipid-mediated interactions. Within catalytic Aur1, we identify a membrane-embedded reaction chamber harboring a conserved H-H-D catalytic triad (H255, H294, and D298) essential for IPC synthesis. Structural comparisons illuminate the mechanism of ceramide recognition and reveal how AbA acts as a competitive inhibitor by occupying the substrate-binding pocket. Further analyses identify key residues involved in AbA binding and explain the molecular basis of drug resistance. Together, these findings advance the mechanistic understanding of fungal IPC biosynthesis and inhibition, and establish a foundation for developing new antifungal drugs targeting IPC synthase. Antifungal aureobasidin A (AbA) targets inositol phosphorylceramide (IPC) synthase. The authors trap its ceramide-bound and drugbound states, revealing the catalytic and drug-inhibition mechanisms, providing basis for antifungal drug development.

N -glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N -glycosylated. The reaction is catalysed by an eight-protein oligosaccharyltransferase (OST) complex that is embedded in the endoplasmic reticulum membrane. Our understanding of eukaryotic protein N -glycosylation has been limited owing to the lack of high-resolution structures. Here we report a 3.5 Å resolution cryo-electron microscopy structure of the Saccharomyces cerevisiae OST complex, revealing the structures of subunits Ost1–Ost5, Stt3, Wbp1 and Swp1. We found that seven phospholipids mediate many of the inter-subunit interactions, and an Stt3 N -glycan mediates interactions with Wbp1 and Swp1 in the lumen. Ost3 was found to mediate the OST–Sec61 translocon interface, funnelling the acceptor peptide towards the OST catalytic site as the nascent peptide emerges from the translocon. The structure provides insights into co-translational protein N -glycosylation, and may facilitate the development of small-molecule inhibitors that target this process. The high-resolution structure of the entire eukaryotic oligosaccharyltransferase complex is determined, revealing the role of membrane lipids in mediating inter-subunit interactions, and the mechanism by which the complex enables protein N -glycosylation. Structure of N -glycosylation enzyme In eukaryotic cells, some 90% of secretory and membrane-bound proteins are modified by gaining a sugar molecule in a process known as N -glycosylation. The enzyme that catalyses this reaction is the oligosaccharyltransferase (OST) complex. Obtaining high-resolution structures of the OST complex would improve understanding of protein N -glycosylation, but this has not been an easy feat as the complex consists of eight protein subunits embedded in the membrane of the endoplasmic reticulum. Huilin Li and colleagues report the cryo-electron microscropy structure of the entire eukaryotic OST complex at a resolution of 3.5 Å. The findings provide insights into the role of membrane lipids in mediating inter-subunit interactions within OST, and the mechanism by which this complex ensures co-translational and co-translocational protein N -glycosylation.

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli- based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N- linked and O -linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities. Cell-free gene expression systems are an attractive platform for biomanufacturing and synthetic biology. Here the authors characterize native membrane vesicles in E. coli extracts for improved glycoengineering.

Asparagine-linked glycosylation is a post-translational modification of proteins containing the conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host–pathogen interactions. The reaction is catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologues in bacteria and archaea. Here we report the X-ray structure of a bacterial OST, the PglB protein of Campylobacter lari , in complex with an acceptor peptide. The structure defines the fold of STT3 proteins and provides insight into glycosylation sequon recognition and amide nitrogen activation, both of which are prerequisites for the formation of the N-glycosidic linkage. We also identified and validated catalytically important, acidic amino acid residues. Our results provide the molecular basis for understanding the mechanism of N-linked glycosylation. Structure of a glycoprotein-producing enzyme More than half of the proteins in eukaryotes are glycoproteins, with specific amino-acid side chains linked to oligosaccharides. The most frequent of these chemical modifications is asparagine-linked glycosylation, catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The X-ray structure of a bacterial OST from Campylobacter lari in complex with an acceptor peptide has now been determined. The structure provides the molecular basis for understanding amide nitrogen activation and glycosylation, and offers opportunities for the production of glycoprotein and glycoconjugate therapeutics.

Human immunoglobulin G (IgG) antibodies are a major class of biotherapeutics and undergo N- linked glycosylation in their Fc domain, which is critical for immune functions and therapeutic activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. Previous attempts to engineer Escherichia coli for this purpose have met limited success due in part to the lack of oligosaccharyltransferase (OST) enzymes that can install N- glycans at the conserved N297 site in the Fc region. Here, we identify a single-subunit OST from Desulfovibrio marinus with relaxed substrate specificity that catalyzes glycosylation of native Fc acceptor sites. By chemoenzymatic remodeling the attached bacterial glycans to homogeneous, asialo complex-type G2 N -glycans, the E. coli -derived Fc binds human FcγRIIIa/CD16a, a key receptor for antibody-dependent cellular cytotoxicity (ADCC). Overall, the discovery of D. marinus OST provides previously unavailable biocatalytic capabilities and sets the stage for using E. coli to produce fully human antibodies. Glycosylation of human IgG antibodies in bacterial hosts has proven challenging. Here, the authors identify a bacterial enzyme enabling E. coli to glycosylate IgGs at native sites, thereby advancing therapeutic antibody development in this host.

Oligosaccharyltransferase (OST) is a multi-span membrane protein complex that catalyzes the addition of glycans to selected Asn residues within nascent polypeptides in the lumen of the endoplasmic reticulum. This process, termed N-glycosylation, is a fundamental post-translational protein modification that is involved in the quality control, trafficking of proteins, signal transduction, and cell-to-cell communication. Given these crucial roles, N-glycosylation is essential for homeostasis at the systemic and cellular levels, and a deficiency in genes that encode for OST subunits often results in the development of complex genetic disorders. A growing body of evidence has also demonstrated that the expression of OST subunits is cell context-dependent and is frequently altered in malignant cells, thus contributing to tumor cell survival and proliferation. Importantly, a recently developed inhibitor of OST has revealed this enzyme as a potential target for the treatment of incurable drug-resistant tumors. This review summarizes our current knowledge regarding the functions of OST in the light of health and tumor progression, and discusses perspectives on the clinical relevance of inhibiting OST as a tumor treatment.

Eukaryotes have an elaborate trafficking and quality-control system for secreted glycoproteins. The glycosylation pathway begins in the endoplasmic reticulum with the enzyme oligosaccharyltransferase (OST), which attaches a long chain of sugars to asparagine residues of target proteins. Wild et al. report a cryo-electron microscopy structure of yeast OST, which includes eight separate membrane proteins. The central catalytic subunit contains binding sites for substrates and is flanked by accessory subunits that may facilitate delivery of newly translocated proteins for glycosylation. Science , this issue p. 545 Accessory subunits stabilize oligosaccharyltransferase and help target substrates for glycosylation. Oligosaccharyltransferase (OST) is an essential membrane protein complex in the endoplasmic reticulum, where it transfers an oligosaccharide from a dolichol-pyrophosphate–activated donor to glycosylation sites of secretory proteins. Here we describe the atomic structure of yeast OST determined by cryo–electron microscopy, revealing a conserved subunit arrangement. The active site of the catalytic STT3 subunit points away from the center of the complex, allowing unhindered access to substrates. The dolichol-pyrophosphate moiety binds to a lipid-exposed groove of STT3, whereas two noncatalytic subunits and an ordered N-glycan form a membrane-proximal pocket for the oligosaccharide. The acceptor polypeptide site faces an oxidoreductase domain in stand-alone OST complexes or is immediately adjacent to the translocon, suggesting how eukaryotic OSTs efficiently glycosylate a large number of polypeptides before their folding.

Inulosucrase from Lactobacillus reuteri 121 (LrInu) exhibits promise in the synthesis of prebiotic inulin and fructooligosaccharides. However, for its use in industry, LrInu’s thermostability is a crucial consideration. In this study, the computational program FireProt was used to predict the thermostable variants of LrInu. Using rational criteria, nine variants were selected for protein expression and characterization. The G237P variant was determined to be the greatest designed candidate due to its greatly enhanced stability and activity in comparison to the wild-type enzyme. The optimum temperature of G237P increased from 50 to 60°C, with an over 5-fold increase in the half-life. Spectroscopy studies revealed that the G237P mutation could prevent the structural change in LrInu caused by heat or urea treatment. Molecular dynamics (MD) simulations showed that the enhanced thermostability of the G237P variant resulted from an increase in structural rigidity and the number of native contacts within the protein molecule. In addition, G237P variant synthesizes inulin with greater efficiency than WT.Key points• Thermostable inulosucrase variant(s) were designed by Fireprot server.• G237P variant showed significantly improved thermostability compared to the wild type.• Inulin is synthesized more efficiently by G237P variant.