Subtle changes in ligand-receptor interactions dramatically alter transcriptional outcomes of pregnane X receptor modulators
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Huber, Andrew D.↗
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Discovery of [cis-3-({(5R)-5-[(7-Fluoro-1,1-dimethyl-2,3-dihydro-1H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5H)-yl}carbonyl)cyclobutyl]acetic Acid (TAK-828F) as a Potent, Selective, and Orally Available Novel Retinoic Acid Receptor-Related Orphan Receptor [gamma]t Inverse Agonist
Abstract not provided
Kono, Mitsunori↗
Intra- and intermolecular atomic-scale interactions in the receptor binding domain of SARS-CoV-2 spike protein: implication for ACE2 receptor binding
Five structural domains in chain A and partial charge distribution in RBD with same orientation as of chain A.
Adhikari, Puja↗
MoS 2 nanosheets effectively bind to the receptor binding domain of the SARS-CoV-2 spike protein and destabilize the spike–human ACE2 receptor interactions
MoS 2 nanosheet holds the potential to bind to the spike protein of SARS CoV-2 and inhibit stable spike–ACE2 interactions.
Chemistry↗
Involvement of multiple scavenger receptors in advanced glycation end product-induced vessel tube formation in endothelial cells
Highlights: • Multiple scavenger receptors contribute to AGE-induced angiogenesis. • RAGE contributes to AGE-induced angiogenesis by upregulation of scavenger receptors. • AGE activates mTOR signaling. Toxic advanced glycation end products (toxic AGEs) derived from glycolaldehyde (AGE3) have been implicated in the development of diabetic vascular complications such as retinopathy characterised by excessive angiogenesis. Different receptor types, such as receptor for AGEs (RAGE), Toll like receptor-4 and scavenger receptors, are expressed in endothelial cells and contribute to AGE-elicited alteration of cell function. In the present study, we examined the involvement of AGE-related receptors on AGE-induced angiogenesis in endothelial cells. The effects of pharmacological inhibitors or receptor neutralizing antibodies on AGE3-induced tube formation were investigated using the in vitro Matrigel tube formation assay in b.End5 cells (mouse endothelial cells). AGE3-induced signalling pathways and receptor expression changes were analysed by Western blot analysis and flow cytometry, respectively. Both FPS-ZM1, a RAGE inhibitor, and fucoidan, a ligand for scavenger receptors, suppressed AGE3-induced tube formation. Cocktails of neutralizing antibodies against the scavenger receptors CD36, CD163 and LOX-1 prevented AGE3-induced tube formation. AGE3 activated mTOR signalling, resulting in facilitation of tube formation. Activation of the AGE-RAGE pathway also led to the upregulation of scavenger receptors. Taken together, our findings suggest that the scavenger receptors CD36, CD163 and LOX-1 in conjunction with the RAGE receptor work together to mediate toxic AGE-induced facilitation of angiogenesis.
60 APPLIED LIFE SCIENCES↗
Structure of the EphB6 receptor ectodomain
Eph receptors are the largest group amongst the receptor tyrosine kinases and are divided into two subgroups, A and B, based on ligand binding specificities and sequence conservation. Through ligand-induced and ligand-independent activities, Ephs play central roles in diverse biological processes, including embryo development, regulation of neuronal signaling, immune responses, vasculogenesis, as well as tumor initiation, progression, and metastasis. The Eph extracellular regions (ECDs) are constituted of multiple domains, and previous structural studies of the A class receptors revealed how they interact with ephrin ligands and simultaneously mediate Eph-Eph clustering necessary for biological activity. Specifically, EphA structures highlighted a model, where clustering of ligand-bound receptors relies on two distinct receptor/receptor interfaces. Interestingly, most unliganded A class receptors also form an additional, third interface, between the ligand binding domain (LBD) and the fibronectin III domain (FN3) of neighboring molecules. Structures of B-class Eph ECDs, on the other hand, have never been reported. To further our understanding of Eph receptor function, we crystallized the EphB6-ECD and determined its three-dimensional structure using X-ray crystallography. EphB6 has important functions in both normal physiology and human malignancies and is especially interesting because this atypical receptor innately lacks kinase activity and our understanding of the mechanism of action is still incomplete. Our structural data reveals the overall EphB6-ECD architecture and shows EphB6-LBD/FN3 interactions similar to those observed for the unliganded A class receptors, suggesting that these unusual interactions are of general importance to the Eph group. We also observe unique structural features, which likely reflect the atypical signaling properties of EphB6, namely the need of co-receptor(s) for this kinase-inactive Eph. These findings provide new valuable information on the structural organization and mechanism of action of the B-class Ephs, and specifically EphB6, which in the future will assist in identifying clinically relevant targets for cancer therapy.
59 BASIC BIOLOGICAL SCIENCES↗
Cloning and Characterization of Aedes aegypti Trypsin Modulating Oostatic Factor (TMOF) Gut Receptor
Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA− was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.
Borovsky, Dov↗
Kainate receptor channel opening and gating mechanism
Kainate receptors, a subclass of ionotropic glutamate receptors, are tetrameric ligand-gated ion channels that mediate excitatory neurotransmission. Kainate receptors modulate neuronal circuits and synaptic plasticity during the development and function of the central nervous system and are implicated in various neurological and psychiatric diseases, including epilepsy, depression, schizophrenia, anxiety and autism. Although structures of kainate receptor domains and subunit assemblies are available, the mechanism of kainate receptor gating remains poorly understood. Here we present cryo-electron microscopy structures of the kainate receptor GluK2 in the presence of the agonist glutamate and the positive allosteric modulators lectin concanavalin A and BPAM344. Concanavalin A and BPAM344 inhibit kainate receptor desensitization and prolong activation by acting as a spacer between the amino-terminal and ligand-binding domains and a stabilizer of the ligand-binding domain dimer interface, respectively. Channel opening involves the kinking of all four pore-forming M3 helices. Our structures reveal the molecular basis of kainate receptor gating, which could guide the development of drugs for treatment of neurological disorders.
59 BASIC BIOLOGICAL SCIENCES↗
Histopathological characteristics of PRRS and expression profiles of viral receptors in the piglet immune system
Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious viral disease that causes significant economic losses to the swine industry worldwide. PRRS virus (PRRSV) infection is a receptor-mediated endocytosis and replication process. The purpose of this study was to determine the localization and expression of four important PRRSV receptors in immunological organs of piglets. After piglets were infected with PRRSV, Hematoxylin and Eosin staining, immunofluorescence, and Western blot were used to perform histopathological examination and receptors distribution analysis. The results showed that PRRSV caused severe damage to the piglets’ immune organs, including atrophy of the thymus and swelling of lymph node. Histopathological lesions were mainly observed in the lung and lymph node and were characterized by interstitial pneumonia, collapsed follicles, exhaustion of germinal centers, and extensive hemorrhage. Immunofluorescence staining and Western blot results showed that the receptors of CD163 and NMHCII-A were mainly distributed in the thymus, hilar lymph nodes, and mesenteric lymph nodes. However, Sn and vimentin receptors were expressed at low levels in the immune organs of piglets. The distribution of the four receptors in the immune organs was more concentrated in the cortex but was more scattered in the medulla. Compared to the control group, the relative expression of the four receptors increased significantly in most immune organs after viral infection. In conclusion, our study examined the distribution and expression of four PRRSV receptors in immunological organs. We observed a significant increase in the expression of Sn, CD163, and vimentin following viral infection. These findings may provide potential targets for future antiviral reagent design or vaccine development.
Chen, Hong↗
Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors
The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.
59 BASIC BIOLOGICAL SCIENCES↗
Update of Receptor Locations for INL National Emissions Standard for Hazardous Air Pollutants (NESHAP) Assessments
This report documents the selection of updated public receptor locations for use in conducting regulatory assessments of radioactive air emissions for Idaho National Laboratory (INL) facilities. The basis for this receptor location analysis is found in 40 CFR 61 Subpart H, “National Emissions Standards for Emissions of Radionuclide Other than Radon from Department of Energy Facilities,” where the requirements for identifying National Emission Standards for Hazardous Air Pollutants (NESHAP) receptor locations are outlined. Receptor locations (residences, schools, businesses, or farm operations) were identified using high-resolution satellite imagery. This assessment identified 31 unique receptor locations for all INL Site facilities, and 30 receptor locations for INL in-town facilities that have the potential to emit radioactive materials. Identification of receptors for each in-town facility is new to this report.
12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
Solvent Acts as the Referee in a Match‐Up Between Charged and Preorganized Receptors
The prevalence of anion-cation contacts in biomolecular recognition under aqueous conditions suggests that ionic interactions should dominate the binding of anions in solvents across both high and low polarities. Investigations of this idea using titrations in low polarity solvents are impaired by interferences from ion pairing that prevent a clear picture of binding. To address this limitation and test the impact of ion-ion interactions across multiple solvents, we quantified chloride binding to a cationic receptor after accounting for ion pairing. In these studies, we created a chelate receptor using aryl-triazole CH donors and a quinolinium unit that directs its cationic methyl inside the binding pocket. In low-polarity dichloromethane, the 1 : 1 complex (log K 1 : 1 ~ 7.3) is more stable than neutral chelates, but fortuitously comparable to a preorganized macrocycle (log K 1 : 1 ~ 6.9). Polar acetonitrile and DMSO diminish stabilities of the charged receptor (log K 1 : 1 ~ 3.7 and 1.9) but surprisingly 100-fold more than the macrocycle. While both receptors lose stability by dielectric screening of electrostatic stability, the cationic receptor also pays additional costs of organization. Thus even though the charged receptor has stronger binding in apolar solvents, the uncharged receptor has more anion affinity in polar solvents.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Spatial organization of FcγR and TLR2/1 on phagosome membranes differentially regulates their synergistic and inhibitory receptor crosstalk
Abstract Many innate immune receptors function collaboratively to detect and elicit immune responses to pathogens, but the physical mechanisms that govern the interaction and signaling crosstalk between the receptors are unclear. In this study, we report that the signaling crosstalk between Fc gamma receptor (FcγR) and Toll-like receptor (TLR)2/1 can be overall synergistic or inhibitory depending on the spatial proximity between the receptor pair on phagosome membranes. Using a geometric manipulation strategy, we physically altered the spatial distribution of FcγR and TLR2 on single phagosomes. We demonstrate that the signaling synergy between FcγR and TLR2/1 depends on the proximity of the receptors and decreases as spatial separation between them increases. However, the inhibitory effect from FcγRIIb on TLR2-dependent signaling is always present and independent of receptor proximity. The overall cell responses are an integration from these two mechanisms. This study presents quantitative evidence that the nanoscale proximity between FcγR and TLR2 functions as a key regulatory mechanism in their signaling crosstalk.
59 BASIC BIOLOGICAL SCIENCES↗
Phenanthroline-strapped calix[4]pyrroles: anion receptors displaying affinity reversal as a function of solvent polarity
Calix[4]pyrroles 1 and 2, diametrically strapped with a phenanthroline via ester and amide linkages, respectively, have been synthesized as anion receptors. It was revealed by 1 H NMR spectroscopic analysis that receptors 1 and 2 possessing both hydrogen bonding donors and acceptors were able to bind the bicarbonate anion (as its tetraethylammonium (TEA + ) or sodium salt) in CDCl 3 , as well as in 15% aqueous DMSO with high affinity and selectivity over other anions. The amide-based receptor 2 contains additional potential hydrogen bonding donors relative to its ester-based congener 1. Nevertheless, in CDCl 3 receptor 1 was found to display a higher affinity for all test anions than receptor 2. In contrast, in 15% aqueous DMSO solution the affinities of receptor 2 for anions, in particular chloride, bicarbonate, and dihydrogen phosphate, were enhanced, whereas those of receptor 1 were reduced dramatically with no appreciable interaction being seen in the case of most test anions considered in this study. These reversals in selectivity and affinities underscore the importance of solvent in regulating the recognition features of seemingly similiar anion binding agents.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Structure-based development of a subtype-selective orexin 1 receptor antagonist
Orexin receptors belong to the superfamily of G-protein–coupled receptors (GPCRs) which represent the largest class of drug targets in humans. Despite the recent progress in structural biology, the development of subtype-selective orexin receptor and GPCR ligands in general remains challenging, due to the high sequence similarity among individual receptor subtypes. However, subtype-selective molecules are key to discerning the individual contributions of receptor subtypes to (patho-)physiology. Starting from the clinically used, non–subtype-selective orexin receptor antagonist suvorexant, we demonstrate how docking, crystallography, medicinal chemistry, and in vitro pharmacology can be combined to exploit single amino acid sequence differences for the discovery of subtype-selective probes. Such compounds will help to better understand orexin receptor pharmacology and develop promising drug candidates.
59 BASIC BIOLOGICAL SCIENCES↗
Pharmacological Profiling of a Brugia malayi Muscarinic Acetylcholine Receptor as a Putative Antiparasitic Target
The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor of B. malayi (Bma-GAR-3) that is highly expressed across intramammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Finally, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.
60 APPLIED LIFE SCIENCES↗
The role of 9-O-acetylated glycan receptor moieties in the typhoid toxin binding and intoxication
Typhoid toxin is an A2B5 toxin secreted from Salmonella Typhi-infected cells during human infection and is suggested to contribute to typhoid disease progression and the establishment of chronic infection. To deliver the enzymatic ‘A’ subunits of the toxin to the site of action in host cells, the receptor-binding ‘B’ subunit PltB binds to the trisaccharide glycan receptor moieties terminated in N-acetylneuraminic acid (Neu5Ac) that is α2–3 or α2–6 linked to the underlying disaccharide, galactose (Gal) and N-acetylglucosamine (GlcNAc). Neu5Ac is present in both unmodified and modified forms, with 9-O-acetylated Neu5Ac being the most common modification in humans. Here we show that host cells associated with typhoid toxin-mediated clinical signs express both unmodified and 9-O-acetylated glycan receptor moieties. We found that PltB binds to 9-O-acetylated α2–3 glycan receptor moieties with a markedly increased affinity, while the binding affinity to 9-O-acetylated α2–6 glycans is only slightly higher, as compared to the affinities of PltB to the unmodified counterparts, respectively. We also present X-ray co-crystal structures of PltB bound to related glycan moieties, which supports the different effects of 9-O-acetylated α2–3 and α2–6 glycan receptor moieties on the toxin binding. Lastly, we demonstrate that the cells exclusively expressing unmodified glycan receptor moieties are less susceptible to typhoid toxin than the cells expressing 9-O-acetylated counterparts, although typhoid toxin intoxicates both cells. These results reveal a fine-tuning mechanism of a bacterial toxin that exploits specific chemical modifications of its glycan receptor moieties for virulence and provide useful insights into the development of therapeutics against typhoid fever.
59 BASIC BIOLOGICAL SCIENCES↗
Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors
Plant cell surface receptors perceive carbohydrate signaling molecules and hereby establish communication with surrounding microbes. Genetic studies have identified two different classes of lysin motif receptor kinases as gatekeepers that together trigger the symbiotic pathway in plants; however, no structural or functional data of the perception mechanisms switching these receptors from resting state into activation is known. In this study, we use structural biology, biochemical, and genetic approaches to demonstrate how the NFP/NFR5 class of lipochitooligosaccharide (LCO) receptors discriminate bacterial symbionts based on a kinetic proofreading mechanism that controls receptor activation and signaling specificity. We show that the LCO binding site can be engineered to support symbiotic functions, which greatly advance future opportunities for receptor engineering in legumes and nonlegumes.
59 BASIC BIOLOGICAL SCIENCES↗