Human cannabinoid type 1 (CB1) G-protein coupled receptor is a potential therapeutic target for obesity. The previously predicted and experimentally validated ensemble of ligand-free conformations of CB1 [Scott, C. E. et al. Protein Sci. 2013 , 22 , 101 - 113 ; Ahn, K. H. et al. Proteins 2013 , 81 , 1304 - 1317] are used here to predict the binding sites for known CB1-selective inverse agonists including rimonabant and its seven known derivatives. This binding pocket, which differs significantly from previously published models, is used to identify 16 novel compounds expected to be CB1 inverse agonists by exploiting potential new interactions. We show experimentally that two of these compounds exhibit inverse agonist properties including inhibition of basal and agonist-induced G-protein coupling activity, as well as an enhanced level of CB1 cell surface localization. This demonstrates the utility of using the predicted binding sites for an ensemble of CB1 receptor structures for designing new CB1 inverse agonists.

The angiopoietin (Ang)-Tie pathway is essential for the proper maturation and remodeling of the vasculature. Despite its importance in disease, the mechanisms that control signal transduction through this pathway are poorly understood. Here, we demonstrate that heparan sulfate glycosaminoglycans (HS GAGs) regulate Ang-Tie signaling through direct interactions with both Ang ligands and Tie1 receptors. HS GAGs formed ternary complexes with Ang1 or Ang4 and Tie2 receptors, resulting in potentiation of endothelial survival signaling. In addition, HS GAGs served as ligands for the orphan receptor Tie1. The HS-Tie1 interaction promoted Tie1-Tie2 heterodimerization and enhanced Tie1 stability within the mature vasculature. Loss of HS-Tie1 binding using CRISPR-Cas9-mediated mutagenesis in vivo led to decreased Tie protein levels, pathway suppression and aberrant retinal vascularization. Together, these results reveal that sulfated glycans use dual mechanisms to regulate Ang-Tie signaling and are important for the development and maintenance of the vasculature.

Electroreduction of carbon dioxide to hydrocarbons and oxygenates on copper involves reduction to a carbon monoxide adsorbate followed by further transformation to hydrocarbons and oxygenates. Simultaneous improvement of these processes over a single reactive site is challenging due to the linear scaling relationship of the binding strength of key intermediates. Herein, we report improved electroreduction of carbon dioxide by exploiting a one-pot tandem catalysis mechanism based on computational and electrochemical investigations. By constructing a well-defined copper-modified silver surface, adsorbed carbon monoxide generated on the silver sites is proposed to migrate to surface copper sites for the subsequent reduction to methane, which is consistent with insights gained from operando attenuated total reflectance surface enhanced infrared absorption spectroscopic investigations. Our results provide a promising approach for designing carbon dioxide electroreduction catalysts to enable one-pot reduction of products beyond carbon monoxide and formate.

Bitter taste is sensed by bitter taste receptors (TAS2Rs) that belong to the G protein-coupled receptor (GPCR) superfamily. In addition to bitter taste perception, TAS2Rs have been reported recently to be expressed in many extraoral tissues and are now known to be involved in health and disease. Despite important roles of TAS2Rs in biological functions and diseases, no crystal structure is available to help understand the signal transduction mechanism or to help develop selective ligands as new therapeutic targets. We report here the three-dimensional structure of the fully activated TAS2R4 human bitter taste receptor predicted using the GEnSeMBLE complete sampling method. This TAS2R4 structure is coupled to the gustducin G protein and to each of several agonists. We find that the G protein couples to TAS2R4 by forming strong salt bridges to each of the three intracellular loops, orienting the activated Gα5 helix of the Gα subunit to interact extensively with the cytoplasmic region of the activated receptor. We find that the TAS2Rs exhibit unique motifs distinct from typical Class A GPCRs, leading to a distinct activation mechanism and a less stable inactive state. This fully activated bitter taste receptor complex structure provides insight into the signal transduction mechanism and into ligand binding to TAS2Rs.

The understanding of G-protein coupled receptors (GPCRs) is undergoing a revolution due to increased information about their signaling and the experimental determination of structures for more than 25 receptors. The availability of at least one receptor structure for each of the GPCR classes, well separated in sequence space, enables an integrated superfamily-wide analysis to identify signatures involving the role of conserved residues, conserved contacts, and downstream signaling in the context of receptor structures. In this study, we align the transmembrane (TM) domains of all experimental GPCR structures to maximize the conserved inter-helical contacts. The resulting superfamily-wide GpcR Sequence-Structure (GRoSS) alignment of the TM domains for all human GPCR sequences is sufficient to generate a phylogenetic tree that correctly distinguishes all different GPCR classes, suggesting that the class-level differences in the GPCR superfamily are encoded at least partly in the TM domains. The inter-helical contacts conserved across all GPCR classes describe the evolutionarily conserved GPCR structural fold. The corresponding structural alignment of the inactive and active conformations, available for a few GPCRs, identifies activation hot-spot residues in the TM domains that get rewired upon activation. Many GPCR mutations, known to alter receptor signaling and cause disease, are located at these conserved contact and activation hot-spot residue positions. The GRoSS alignment places the chemosensory receptor subfamilies for bitter taste (TAS2R) and pheromones (Vomeronasal, VN1R) in the rhodopsin family, known to contain the chemosensory olfactory receptor subfamily. The GRoSS alignment also enables the quantification of the structural variability in the TM regions of experimental structures, useful for homology modeling and structure prediction of receptors. Furthermore, this alignment identifies structurally and functionally important residues in all human GPCRs. These residues can be used to make testable hypotheses about the structural basis of receptor function and about the molecular basis of disease-associated single nucleotide polymorphisms.

Fasting glucose and insulin are intermediate traits for type 2 diabetes. Here we explore the role of coding variation on these traits by analysis of variants on the HumanExome BeadChip in 60,564 non-diabetic individuals and in 16,491 T2D cases and 81,877 controls. We identify a novel association of a low-frequency nonsynonymous SNV in GLP1R (A316T; rs10305492; MAF=1.4%) with lower FG (β=-0.09±0.01 mmol l(-1), P=3.4 × 10(-12)), T2D risk (OR[95%CI]=0.86[0.76-0.96], P=0.010), early insulin secretion (β=-0.07±0.035 pmolinsulin mmolglucose(-1), P=0.048), but higher 2-h glucose (β=0.16±0.05 mmol l(-1), P=4.3 × 10(-4)). We identify a gene-based association with FG at G6PC2 (pSKAT=6.8 × 10(-6)) driven by four rare protein-coding SNVs (H177Y, Y207S, R283X and S324P). We identify rs651007 (MAF=20%) in the first intron of ABO at the putative promoter of an antisense lncRNA, associating with higher FG (β=0.02±0.004 mmol l(-1), P=1.3 × 10(-8)). Our approach identifies novel coding variant associations and extends the allelic spectrum of variation underlying diabetes-related quantitative traits and T2D susceptibility.

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

Amorphous carbon systems are emerging to have unparalleled properties at multiple length scales, making them the preferred choice for creating advanced materials in many sectors, but the lack of long-range order makes it difficult to establish structure/property relationships. We propose an original computational approach to predict the morphology of carbonaceous materials for arbitrary densities that we apply here to graphitic phases at low densities from 1.15 to 0.16 g/cm3, including glassy carbon. This approach, dynamic reactive massaging of the potential energy surface (DynReaxMas), uses the ReaxFF reactive force field in a simulation protocol that combines potential energy surface (PES) transformations with global optimization within a multidescriptor representation. DynReaxMas enables the simulation of materials synthesis at temperatures close to experiment to correctly capture the interplay of activated vs entropic processes and the resulting phase morphology. We then show that DynReaxMas efficiently and semiautomatically produces atomistic configurations that span wide relevant regions of the PES at modest computational costs. Indeed, we find a variety of distinct phases at the same density, and we illustrate the evolution of competing phases as a function of density ranging from uniform vs bimodal distributions of pore sizes at higher and intermediate density (1.15 g/cm3 and 0.50 g/cm3) to agglomerated vs sparse morphologies, further partitioned into boxed vs hollow fibrillar morphologies, at lower density (0.16 g/cm3). Our observations of diverse phases at the same density agree with experiment. Some of our identified phases provide descriptors consistent with available experimental data on local density, pore sizes, and HRTEM images, showing that DynReaxMas provides a systematic classification of the complex field of amorphous carbonaceous materials that can provide 3D structures to interpret experimental observations.

Understanding the roles of intermediate states in signaling is pivotal to unraveling the activation processes of G protein-coupled receptors (GPCRs). However, the field is still struggling to define these conformational states with sufficient resolution to study their individual functions. Here, we demonstrate the feasibility of enriching the populations of discrete states via conformation-biased mutants. These mutants adopt distinct distributions among five states that lie along the activation pathway of adenosine A2A receptor (A2AR), a class A GPCR. Our study reveals a structurally conserved cation-π lock between transmembrane helix VI (TM6) and Helix8 that regulates cytoplasmic cavity opening as a "gatekeeper" for G protein penetration. A GPCR activation process based on the well-discerned conformational states is thus proposed, allosterically micro-modulated by the cation-π lock and a previously well-defined ionic interaction between TM3 and TM6. Intermediate-state-trapped mutants will also provide useful information in relation to receptor-G protein signal transduction.

According to the World Health Organization (WHO), almost 2 billion people each year are infected worldwide with flu-like pathogens including influenza. This is a contagious disease caused by viruses belonging to the family Orthomyxoviridae. Employee absenteeism caused by flu infection costs hundreds of millions of dollars every year. To successfully treat influenza virus infections, detection of the virus during the initial development phase of the infection is critical, when tens to hundreds of virus-associated molecules are present in the patient's pharynx. In this study, we describe a novel universal diamond biosensor, which enables the specific detection of the virus at ultralow concentrations, even before any clinical symptoms arise. A diamond electrode is surface-functionalized with polyclonal anti-M1 antibodies, which then serve to identify the universal biomarker for the influenza virus, M1 protein. The absorption of the M1 protein onto anti-M1 sites of the electrode change its electrochemical impedance spectra. We achieved a limit of detection of 1 fg/ml in saliva buffer for the M1 biomarker, which corresponds to 5-10 viruses per sample in 5 minutes. Furthermore, the universality of the assay was confirmed by analyzing different strains of influenza A virus.

The last decade has witnessed dramatic growth in the number of reactions catalyzed by electrophilic gold complexes. While proposed mechanisms often invoke the intermediacy of gold-stabilized cationic species, the nature of bonding in these intermediates remains unclear. Herein, we propose that the carbon-gold bond in these intermediates is comprised of varying degrees of both sigma and pi-bonding; however, the overall bond order is generally less than or equal to unity. The bonding in a given gold-stabilized intermediate, and the position of this intermediate on a continuum ranging from gold-stabilized singlet carbene to gold-coordinated carbocation, is dictated by the carbene substituents and the ancillary ligand. Experiments show that the correlation between bonding and reactivity is reflected in the yield of gold-catalyzed cyclopropanation reactions.

Harnessing renewable electricity to drive the electrochemical reduction of CO2 is being intensely studied for sustainable fuel production and as a means for energy storage. Copper is the only monometallic electrocatalyst capable of converting CO2 to value-added products, e.g., hydrocarbons and oxygenates, but suffers from poor selectivity and mediocre activity. Multiple oxidative treatments have shown improvements in the performance of copper catalysts. However, the fundamental underpinning for such enhancement remains controversial. Here, we combine reactivity, in-situ surface-enhanced Raman spectroscopy, and computational investigations to demonstrate that the presence of surface hydroxyl species by co-electrolysis of CO2 with low concentrations of O2 can dramatically enhance the activity of copper catalyzed CO2 electroreduction. Our results indicate that co-electrolysis of CO2 with an oxidant is a promising strategy to introduce catalytically active species in electrocatalysis.

Since Paul Ehrlich's introduction of the "magic bullet" concept in 1908, drug developers have been seeking new ways to target drug activity to diseased cells while limiting effects on normal tissues. In recent years, it has been proposed that coupling riboswitches capable of detecting RNA biomarkers to small interfering RNAs (siRNAs) to create siRNA pro-drugs could selectively activate RNA interference (RNAi) activity in specific cells. However, this concept has not been achieved previously. We report here that we have accomplished this goal, validating a simple and programmable new design that functions reliably in mammalian cells. We show that these conditionally activated siRNAs (Cond-siRNAs) can switch RNAi activity against different targets between clearly distinguished OFF and ON states in response to different cellular RNA biomarkers. Notably, in a rat cardiomyocyte cell line (H9C2), one version of our construct demonstrated biologically meaningful inhibition of a heart-disease-related target gene protein phosphatase 3 catalytic subunit alpha (PPP3CA) in response to increased expression of the pathological marker atrial natriuretic peptide (NPPA) messenger RNA (mRNA). Our results demonstrate the ability of synthetic riboswitches to regulate gene expression in mammalian cells, opening a new path for development of programmable siRNA pro-drugs.

G protein-coupled receptors display multifunctional signaling, offering the potential for agonist structures to promote conformational selectivity for biased outputs. For β2-adrenergic receptors (β2AR), unbiased agonists stabilize conformation(s) that evoke coupling to Gαs (cyclic adenosine monophosphate [cAMP] production/human airway smooth muscle [HASM] cell relaxation) and β-arrestin engagement, the latter acting to quench Gαs signaling, contributing to receptor desensitization/tachyphylaxis. We screened a 40-million-compound scaffold ranking library, revealing unanticipated agonists with dihydroimidazolyl-butyl-cyclic urea scaffolds. The S-stereoisomer of compound C1 shows no detectable β-arrestin engagement/signaling by four methods. However, C1-S retained Gαs signaling-a divergence of the outputs favorable for treating asthma. Functional studies with two models confirmed the biasing: β2AR-mediated cAMP signaling underwent desensitization to the unbiased agonist albuterol but not to C1-S, and desensitization of HASM cell relaxation was observed with albuterol but not with C1-S These HASM results indicate biologically pertinent biasing of C1-S, in the context of the relevant physiologic response, in the human cell type of interest. Thus, C1-S was apparently strongly biased away from β-arrestin, in contrast to albuterol and C5-S C1-S structural modeling and simulations revealed binding differences compared with unbiased epinephrine at transmembrane (TM) segments 3,5,6,7 and ECL2. C1-S (R2 = cyclohexane) was repositioned in the pocket such that it lost a TM6 interaction and gained a TM7 interaction compared with the analogous unbiased C5-S (R2 = benzene group), which appears to contribute to C1-S biasing away from β-arrestin. Thus, an agnostic large chemical-space library identified agonists with receptor interactions that resulted in relevant signal splitting of β2AR actions favorable for treating obstructive lung disease.

Preventing spontaneous crystallization of supersaturated solutions by additives is of critical interest to successful process design and implementation, with numerous applications in chemical, pharmaceutical, medical, pigment, and food industries, but challenges remain in laboratory and industry settings and fundamental understanding is lacking. When copresented with antifreeze proteins (AFPs), otherwise spontaneously crystallizing osmolytes are maintained at high supersaturations for months in over-wintering organisms. Thus, we here explore the inhibition phenomenon by AFPs, using persistent crystallization of a common sugar alcohol, D-mannitol, as a case study. We report experimentally that DAFP1, an insect AFP, completely inhibits D-mannitol nucleation. Computer simulations reveal a new mechanism for crystallization inhibition where the population of the crystal-forming conformers are selectively bound and randomized in solution by hydrogen bonding to the protein surface. These results highlight the advantages of using natural polymers to address crystallization inhibition challenges and suggest new strategies in controlling the nucleation processes.

Macrocycle peptides are promising constructs for imaging and inhibiting extracellular, and cell membrane proteins, but their use for targeting intracellular proteins is typically limited by poor cell penetration. We report the development of a cell-penetrant high-affinity peptide ligand targeted to the phosphorylated Ser474 epitope of the (active) Akt2 kinase. This peptide can function as an allosteric inhibitor, an immunoprecipitation reagent, and a live cell immunohistochemical staining reagent. Two cell penetrant stereoisomers were prepared and shown to exhibit similar target binding affinities and hydrophobic character but 2-3-fold different rates of cell penetration. Experimental and computational studies resolved that the ligands' difference in cell penetration could be assigned to their differential interactions with cholesterol in the membrane. These results expand the tool kit for designing new chiral-based cell-penetrant ligands.

During formation of the Hedgehog (Hh) signaling proteins, cooperative activities of the Hedgehog INTein (Hint) fold and Sterol Recognition Region (SRR) couple autoproteolysis to cholesterol ligation. The cholesteroylated Hh morphogens play essential roles in embryogenesis, tissue regeneration, and tumorigenesis. Despite the centrality of cholesterol in Hh function, the full structure of the Hint-SRR ("Hog") domain that attaches cholesterol to the last residue of the active Hh morphogen remains enigmatic. In this work, we combine molecular dynamics simulations, photoaffinity crosslinking, and mutagenesis assays to model cholesterolysis intermediates in the human Sonic Hedgehog (hSHH) protein. Our results provide evidence for a hydrophobic Hint-SRR interface that forms a dynamic, non-covalent cholesterol-Hog complex. Using these models, we suggest a unified mechanism by which Hh proteins can recruit, sequester, and orient cholesterol, and offer a molecular basis for the effects of disease-causing hSHH mutations.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in β-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was β-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited β-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of β-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the β-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Macroscopic electric motors continue to have a large impact on almost every aspect of modern society. Consequently, the effort towards developing molecular motors1-3 that can be driven by electricity could not be more timely. Here we describe an electric molecular motor based on a [3]catenane4,5, in which two cyclobis(paraquat-p-phenylene)6 (CBPQT4+) rings are powered by electricity in solution to circumrotate unidirectionally around a 50-membered loop. The constitution of the loop ensures that both rings undergo highly (85%) unidirectional movement under the guidance of a flashing energy ratchet7,8, whereas the interactions between the two rings give rise to a two-dimensional potential energy surface (PES) similar to that shown by FOF1 ATP synthase9. The unidirectionality is powered by an oscillating10 voltage11,12 or external modulation of the redox potential13. Initially, we focused our attention on the homologous [2]catenane, only to find that the kinetic asymmetry was insufficient to support unidirectional movement of the sole ring. Accordingly, we incorporated a second CBPQT4+ ring to provide further symmetry breaking by interactions between the two mobile rings. This demonstration of electrically driven continual circumrotatory motion of two rings around a loop in a [3]catenane is free from the production of waste products and represents an important step towards surface-bound14 electric molecular motors.