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Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp 2-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp 3-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.

Puromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 ‘WY’ conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 µM binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1and UGGT2 activity at concentrations higher than 750 µM in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N -glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N -glycan.Significance StatementWhen a candidate drug target is the product of a housekeeping gene - i.e. it is important for the normal functioning of the healthy cell – availability of inhibitors for tests and assays is of paramount importance. One such housekeeping protein is UGGT, the enzyme that makes sure that only correctly folded glycoproteins can leave the endoplasmic reticulum for further trafficking through the secretory pathway. UGGT is a potential drug target against viruses, in certain instances of congenital rare disease, and against some cancers, but no UGGT inhibitors are known yet. We discovered and describe here a small molecule that binds human UGGT1 in vitro and inhibits both isoforms of human UGGT in cellula. The compound paves the way to testing of UGGT inhibition as a potential pharmacological strategy in a number of medical contexts.

Regulation of type-I interferon (IFN) production is essential to the balance between antimicrobial defence and autoimmune disorders. The human protein-coding gene ILRUN (inflammation and lipid regulator with UBA-like and NBR1-like domains, previously C6orf106) was recently characterised as an inhibitor of antiviral and proinflammatory cytokine (interferon-alpha/beta and tumor necrosis factor alpha) transcription. Currently there is a paucity of information about the molecular characteristics of ILRUN, despite it being associated with several diseases including virus infection, coronary artery disease, obesity and cancer. Here, we characterise ILRUN as a highly phylogenetically conserved protein containing UBA-like and a NBR1-like domains that are both essential for inhibition of type-I interferon and tumor necrosis factor alpha) transcription in human cells. We also solved the crystal structure of the NBR1-like domain, providing insights into its potential role in ILRUN function. This study provides critical information for future investigations into the role of ILRUN in health and disease., Immunolgy; Inflammation; Infectious disease; Immune Response; Viruses; Biochemistry; Biomolecules; ILRUN; C6orf106; innate immunity; interferon; p300; CBP

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.

Targeting the host-cell endoplasmic reticulum quality control (ERQC) pathway is an effective broad-spectrum antiviral strategy. The two ER resident α-glucosidases whose sequential action permits entry in this pathway are the targets of glucomimetic inhibitors. Knowledge of the molecular details of the ER α-glucosidase II (α-Glu II) structure was limited. We determined crystal structures of a trypsinolytic fragment of murine α-Glu II, alone and in complex with key catalytic cycle ligands, and four different broad-spectrum antiviral iminosugar inhibitors, two of which are currently in clinical trials against dengue fever. The structures highlight novel portions of the enzyme outside its catalytic pocket which contribute to its activity and substrate specificity. These crystal structures and hydrogen-deuterium exchange mass spectrometry of the murine ER alpha glucosidase II heterodimer uncover the quaternary arrangement of the enzyme's α- and β-subunits, and suggest a conformational rearrangement of ER α-Glu II upon association of the enzyme with client glycoproteins.

International audience; The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

Iminosugars have therapeutic potential against a range of diseases, due to their efficacy as glycosidase inhibitors. A major challenge in the development of iminosugar drugs lies in making a compound that is selective for the glycosidase associated with a given disease. We report the synthesis of ToP-DNJ, an antiviral iminosugar–tocopherol conjugate. Tocopherol was incorporated into the design of the iminosugar in order to direct the drug to the liver and immune cells, specific tissues of interest for antiviral therapy. ToP-DNJ inhibits ER α-glucosidase II at low micromolar concentrations and selectively accumulates in the liver in vivo. In cellular assays, the drug showed efficacy exclusively in immune cells of the myeloid lineage. Taken together, these data demonstrate that inclusion of a native metabolite into an iminosugar provides selectivity with respect to target enzyme, target cell, and target tissue.

Glycoproteins traversing the eukaryotic secretory pathway begin life in the endoplasmic reticulum (ER), where their folding is surveyed by the 170 kDa UDP-glucose:glycoprotein glucosyltransferase (UGGT). The enzyme acts as the single glycoprotein folding quality control checkpoint: it selectively re-glucosylates misfolded glycoproteins, promotes their association with ER lectins and associated chaperones and prevents premature secretion from the ER. UGGT has long resisted structural determination and sequence-based domain boundary prediction. Questions remain on how this single enzyme can flag misfolded glycoproteins of different sizes and shapes for ER-retention, and how it can span variable distances between the site of misfold and a glucose-accepting N-linked glycan on the same glycoprotein. Here, the first crystal structures of a full length eukaryotic UGGT reveal four thioredoxin-like (TRXL) domains arranged in a long arc, which terminates in two β–sandwiches tightly clasping the glucosyltransferase domain. The fold of the molecule is topologically complex, with the first β–sandwich and the fourth TRXL domain being encoded by non-consecutive stretches of sequence. In addition to the crystal structures, a 15 Å cryo-EM reconstruction reveals inter-domain flexibility of the TRXL domains. Double cysteine point mutants that engineer extra inter-domain disulfide bridges rigidify the UGGT structure and exhibit impaired activity. The intrinsic flexibility of the TRXL domains of UGGT may therefore endow the enzyme with the promiscuity needed to recognize and re-glucosylate its many different substrates, and/or enable re-glucosylation of N-linked glycans situated at variable distances from the site of misfold.

The biosynthesis of enveloped viruses depends heavily on the host cell endoplasmic reticulum (ER) glycoprotein quality control (QC) machinery. This dependency exceeds the dependency of host glycoproteins, offering a window for the targeting of ERQC for the development of broad-spectrum antivirals. We determined smallangle X-ray scattering (SAXS) and crystal structures of themain ERQC enzyme, ER alpha-glucosidase II (alpha-GluII; from mouse), alone and in complex with key ligands of its catalytic cycle and antiviral iminosugars, including two that are in clinical trials for the treatment of dengue fever. The SAXS data capture the enzyme's quaternary structure and suggest a conformational rearrangement is needed for the simultaneous binding of a monoglucosylated glycan to both subunits. The X-ray structures with key catalytic cycle intermediates highlight that an insertion between the + 1 and + 2 subsites contributes to the enzyme's activity and substrate specificity, and reveal that the presence of D-mannose at the + 1 subsite renders the acid catalyst less efficient during the cleavage of the monoglucosylated substrate. The complexes with iminosugar antivirals suggest that inhibitors targeting a conserved ring of aromatic residues between the alpha-GluII + 1 and + 2 subsites would have increased potency and selectivity, thus providing a template for further rational drug design.

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

SummaryPotassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.PaperFlick

Effective use of adenovirus-5 (Ad5) in cancer therapy is heavily dependent on the degree to which the virus's natural tropism can be subverted to one that favours tumour cells. This is normally achieved through either engineering of the viral fiber knob or the use of bispecific adaptors that display both adenovirus and tumour antigen receptors. One of the main limitations of these strategies is the need to tailor each engineering event to any given tumour antigen. Here, we explore bispecific adaptors that can utilise established anti-cancer therapeutic antibodies. Conjugates containing bacterially derived antibody binding motifs are efficient at retargeting virus to antibody targets. Here, we develop a humanized strategy whereby we synthesise a re-targeting adaptor based on a chimeric Ad5 ligand/antibody receptor construct. This adaptor acts as a molecular bridge analogous to therapeutic antibody mediated cross-linking of cytotoxic effector and tumour cells during immunotherapy. As a proof or principle, we demonstrate how this adaptor allows efficient viral recognition and entry into carcinoma cells through the therapeutic monoclonal antibodies Herceptin/trastuzumab and bavituximab. We show that targeting can be augmented by use of contemporary antibody enhancement strategies such as the selective elimination of competing serum IgG using "receptor refocusing" enzymes and we envisage that further improvements are achievable by enhancing the affinities between the adaptor and its ligands. Humanized bispecific adaptors offer the promise of a versatile retargeting technology that can exploit both clinically approved adenovirus and therapeutic antibodies.