Structural characterization of binding mode of smoking cessation drugs to nicotinic acetylcholine receptors through study of ligand complexes with acetylcholine-binding protein

Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4β2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.     

Structural basis of digoxin that antagonizes RORgamma t receptor activity and suppresses Th17 cell differentiation and interleukin (IL)-17 production

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4⁺ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.     

A Conserved Mechanism of GABA Binding and Antagonism Is Revealed by Structure-Function Analysis of the Periplasmic Binding Protein Atu2422 in Agrobacterium tumefaciens

Bacterial periplasmic binding proteins (PBPs) and eukaryotic PBP-like domains (also called as Venus flytrap modules) of G-protein-coupled receptors are involved in extracellular GABA perception. We investigated the structural and functional basis of ligand specificity of the PBP Atu2422, which is implicated in virulence and transport of GABA in the plant pathogen Agrobacterium tumefaciens. Five high-resolution x-ray structures of Atu2422 liganded to GABA, Pro, Ala, and Val and of point mutant Atu2422-F77A liganded to Leu were determined. Structural analysis of the ligand-binding site revealed two essential residues, Phe⁷⁷ and Tyr²⁷⁵, the implication of which in GABA signaling and virulence was confirmed using A. tumefaciens cells expressing corresponding Atu2422 mutants. Phe⁷⁷ restricts ligand specificity to α-amino acids with a short lateral chain, which act as antagonists of GABA signaling in A. tumefaciens. Tyr²⁷⁵ specifically interacts with the GABA γ-amino group. Conservation of these two key residues in proteins phylogenetically related to Atu2422 brought to light a subfamily of PBPs in which all members could bind GABA and short α-amino acids. This work led to the identification of a fingerprint sequence and structural features for defining PBPs that bind GABA and its competitors and revealed their occurrence among host-interacting proteobacteria.     

Glucose and insulin induction of bile acid synthesis: mechanisms and implication in diabetes and obesity

Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7α-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.     

Structural and mechanistic insights into the interaction of cytochrome P4503A4 with bromoergocryptine, a type I ligand

Cytochrome P4503A4 (CYP3A4), a major human drug-metabolizing enzyme, is responsible for the oxidation and clearance of the majority of administered drugs. One of the CYP3A4 substrates is bromoergocryptine (BEC), a dopamine receptor agonist prescribed for the inhibition of prolactin secretion and treatment of Parkinson disease, type 2 diabetes, and several other pathological conditions. Here we present a 2.15 Å crystal structure of the CYP3A4-BEC complex in which the drug, a type I heme ligand, is bound in a productive mode. The manner of BEC binding is consistent with the in vivo metabolite analysis and identifies the 8′ and 9′ carbons of the proline ring as the primary sites of oxidation. The crystal structure predicts the importance of Arg²¹² and Thr²²⁴ for binding of the tripeptide and lysergic moieties of BEC, respectively, which we confirmed experimentally. Our data support a three-step BEC binding model according to which the drug binds first at a peripheral site without perturbing the heme spectrum and then translocates into the active site cavity, where formation of a hydrogen bond between Thr²²⁴ and the N1 atom of the lysergic moiety is followed by a slower conformational readjustment of the tripeptide group modulated by Arg²¹².     

Structure and function of the RedJ protein, a thioesterase from the prodiginine biosynthetic pathway in Streptomyces coelicolor

Prodiginines are a class of red-pigmented natural products with immunosuppressant, anticancer, and antimalarial activities. Recent studies on prodiginine biosynthesis in Streptomyces coelicolor have elucidated the function of many enzymes within the pathway. However, the function of RedJ, which was predicted to be an editing thioesterase based on sequence similarity, is unknown. We report here the genetic, biochemical, and structural characterization of the redJ gene product. Deletion of redJ in S. coelicolor leads to a 75% decrease in prodiginine production, demonstrating its importance for prodiginine biosynthesis. RedJ exhibits thioesterase activity with selectivity for substrates having long acyl chains and lacking a β-carboxyl substituent. The thioesterase has 1000-fold greater catalytic efficiency with substrates linked to an acyl carrier protein (ACP) than with the corresponding CoA thioester substrates. Also, RedJ strongly discriminates against the streptomycete ACP of fatty acid biosynthesis in preference to RedQ, an ACP of the prodiginine pathway. The 2.12 Å resolution crystal structure of RedJ provides insights into the molecular basis for the observed substrate selectivity. A hydrophobic pocket in the active site chamber is positioned to bind long acyl chains, as suggested by a long-chain ligand from the crystallization solution bound in this pocket. The accessibility of the active site is controlled by the position of a highly flexible entrance flap. These data combined with previous studies of prodiginine biosynthesis in S. coelicolor support a novel role for RedJ in facilitating transfer of a dodecanoyl chain from one acyl carrier protein to another en route to the key biosynthetic intermediate 2-undecylpyrrole.     

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with K_b values of 21–63 nm at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.     

Crystal structure of the largest subunit of a bacterial RNA-guided immune complex and its role in DNA target binding

Prokaryotes make use of small RNAs encoded by CRISPR (clustered regularly interspaced short palindromic repeat) loci to provide immunity against bacteriophage or plasmid invasion. In Escherichia coli, the CRISPR-associated complex for antiviral defense (Cascade) utilizes these RNAs to target foreign DNA for destruction. CasA, the largest subunit of Cascade, is essential for its function. Here we report the crystal structure of Thermus thermophilus CasA. The structure is composed of two domains that are arranged in a chair-like conformation with a novel fold forming the larger N-terminal domain. Docking of the crystal structure into cryo-electron microscopy maps reveals two loops in CasA that likely have important functions in DNA target binding. Finally, DNA binding experiments show that CasA is essential for binding of Cascade to DNA target.     

Caenorhabditis elegans mom-4 is required for the activation of the p38 MAPK signaling pathway in the response to Pseudomonas aeruginosa infection

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. aeruginosa.     

The Role of a Novel Auxiliary Pocket in Bacterial Phenylalanyl-tRNA Synthetase Druggability

The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.     

Biochemical and Structural Insights into RNA Binding by Ssh10b,a Member of the Highly Conserved Sac10b Protein Family in Archaea

Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular β-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.     

Structural Insights into the Unusually Strong ATPase Activity of the AAA Domain of the Caenorhabditis elegans Fidgetin-like 1 (FIGL-1) Protein

The FIGL-1 (fidgetin like-1) protein is a homolog of fidgetin, a protein whose mutation leads to multiple developmental defects. The FIGL-1 protein contains an AAA (ATPase associated with various activities) domain and belongs to the AAA superfamily. However, the biological functions and developmental implications of this protein remain unknown. Here, we show that the AAA domain of the Caenorhabditis elegans FIGL-1 protein (CeFIGL-1-AAA), in clear contrast to homologous AAA domains, has an unusually high ATPase activity and forms a hexamer in solution. By determining the crystal structure of CeFIGL-1-AAA, we found that the loop linking helices α9 and α10 folds into the short helix α9a, which has an acidic surface and interacts with a positively charged surface of the neighboring subunit. Disruption of this charge interaction by mutagenesis diminishes both the ATPase activity and oligomerization capacity of the protein. Interestingly, the acidic residues in helix α9a of CeFIGL-1-AAA are not conserved in other homologous AAA domains that have relatively low ATPase activities. These results demonstrate that the sequence of CeFIGL-1-AAA has adapted to establish an intersubunit charge interaction, which contributes to its strong oligomerization and ATPase activity. These unique properties of CeFIGL-1-AAA distinguish it from other homologous proteins, suggesting that CeFIGL-1 may have a distinct biological function.