γ-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, and disturbances in the GABAergic system have been implicated in numerous neurological and neuropsychiatric diseases. The GABAB receptor is a heterodimeric class C G protein-coupled receptor (GPCR) consisting of GABAB1a/b and GABAB2 subunits. Two GABAB receptor ligand binding sites have been described, namely the orthosteric GABA binding site located in the extracellular GABAB1 Venus fly trap domain and the allosteric binding site found in the GABAB2 transmembrane domain. To date, the only experimentally solved three-dimensional structures of the GABAB receptor are of the Venus fly trap domain. GABAB receptor allosteric modulators, however, show great therapeutic potential, and elucidating the structure of the GABAB2 transmembrane domain may lead to development of novel drugs and increased understanding of the allosteric mechanism of action. Despite the lack of x-ray crystal structures of the GABAB2 transmembrane domain, multiple crystal structures belonging to other classes of GPCRs than class A have been released within the last years. More closely related template structures are now available for homology modelling of the GABAB receptor. Here, multiple homology models of the GABAB2 subunit of the GABAB receptor have been constructed using templates from class A, B and C GPCRs, and docking of five clusters of positive allosteric modulators and decoys has been undertaken to select models that enrich the active compounds. Using this ligand-guided approach, eight GABAB2 homology models have been chosen as possible structural representatives of the transmembrane domain of the GABAB2 subunit. To the best of our knowledge, the present study is the first to describe homology modelling of the transmembrane domain of the GABAB2 subunit and the docking of positive allosteric modulators in the receptor.
In the Carbohydrate-Active Enzyme (CAZy) database, glycoside hydrolase family 5 (GH5) is a large family with more than 6,000 sequences. Among the 51 described GH5 subfamilies, subfamily GH5_26 contains members that display either endo-(1,4)-glucanase or (1,3;1,4)-glucanase activities. In this study, we focused on the GH5_26 enzyme from Saccharophagus degradans (SdGluc5_26A), a marine bacterium known for its capacity to degrade a wide diversity of complex polysaccharides. SdGluc5_26A displays lichenase activity toward (1,3; 1,4)-glucans with a side cellobiohydrolase activity toward (1, 4)-glucans. The three-dimensional structure of SdGluc5_26A adopts a stable trimeric quaternary structure also observable in solution. The N-terminal region of SdGluc5_26A protrudes into the active site of an adjacent monomer. To understand whether this occupation of the active site could influence its activity, we conducted a comprehensive enzymatic characterization of SdGluc5_26A and of a mutant truncated at the N terminus. Ligand complex structures and kinetic analyses reveal that the N terminus governs the substrate specificity of SdGluc5_26A. Its deletion opens the enzyme cleft at the ؊3 subsite and turns the enzyme into an endo-(1,4)-glucanase. This study demonstrates that experimental approaches can reveal structure-function relationships out of reach of current bioinformatic predictions.
N‐methyl‐ d ‐aspartate receptors ( NMDAR ) are widely expressed in the brain. GluN2B subunit‐containing NMDAR s has recently attracted significant attention as potential pharmacological targets, with emphasis on the functional properties of allosteric antagonists. We used primary cultures from chicken embryo forebrain (E10), expressing native GluN2B‐containing NMDA receptors as a novel model system. Comparing the inhibition of calcium influx by well‐known GluN2B subunit‐specific allosteric antagonists, the following rank order of potency was found: EVT ‐101 ( EC 50 22 ± 8 nmol/L) > Ro 25‐6981 ( EC 50 60 ± 30 nmol/L) > ifenprodil ( EC 50 100 ± 40 nmol/L) > eliprodil ( EC 50 1300 ± 700 nmol/L), similar to previous observations in rat cortical cultures and cell lines overexpressing chimeric receptors. The less explored Ro 04‐5595 had an EC 50 of 186 ± 32 nmol/L. Venturing to explain the differences in potency, binding properties were further studied by in silico docking and molecular dynamics simulations using x‐ray crystal structures of GluN1/GluN2B amino terminal domain. We found that Ro 04‐5595 was predicted to bind the recently discovered EVT ‐101 binding site, not the ifenprodil‐binding site. The EVT ‐101 binding pocket appears to accommodate more structurally different ligands than the ifenprodil‐binding site, and contains residues essential in ligand interactions necessary for calcium influx inhibition. For the ifenprodil site, the less effective antagonist (eliprodil) fails to interact with key residues, while in the EVT ‐101 pocket, difference in potency might be explained by differences in ligand‐receptor interaction patterns.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.