Innate immune recognition of single-stranded RNA via Toll-like receptor 7 (TLR7) plays an important regulatory role in the development of the immune response. In the study, we used PCR-single-strand ...conformational polymorphism (PCR-SSCP) analysis to investigate genetic variations in the sequence encoding the ectodomain (ECD) of ovine TLR7. PCR was carried out using 12 different primer pairs (ECD-1– ECD-12), and polymorphisms were detected in the products for 4/12 pairs (ECD-2, -5, -6, and -7). ECD-2 and ECD -5 amplified products with mutations in Suffolk and Kazakstan sheep, while ECD-6 and ECD-7 amplified products with mutations only exist in Suffolk. The second pair of primers mutation in Suffolk does not conform to Hardy Weinberg equilibrium. As the mutations identified in this study may influence the structure and function of TLR7 and may thus affect the immune response to pathogens. The data indicate to undertake further disease associations studies to examine the usefulness of these findings in livestock breeding.
Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca(2+) homeostasis through the regulation of parathyroid hormone secretion. It functions as a ...disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca(2+) and PO4(3-) ions. Both ions are crucial for structural integrity of the receptor. While Ca(2+) ions stabilize the active state, PO4(3-) ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.
Cell adhesion between oligodendrocytes and neuronal axons is a critical step for myelination that enables the rapid propagation of action potential in the central nervous system. Here, we show that ...the transmembrane protein teneurin-4 plays a role in the cell adhesion required for the differentiation of oligodendrocytes. We found that teneurin-4 formed molecular complexes with all of the four teneurin family members and promoted cell-cell adhesion. Oligodendrocyte lineage cells attached to the recombinant extracellular domain of all the teneurins and formed well-branched cell processes. In an axon-mimicking nanofibers assay, nanofibers coated with the recombinant teneurin-4 extracellular domain increased the differentiation of oligodendrocytes. Our results show that teneurin-4 binds to all teneurins through their extracellular domain, which facilitates the oligodendrocyte-axon adhesion, and promotes oligodendrocyte differentiation via its homophilic interaction.
•Teneurin-4 forms molecular complexes with all teneurin family members.•Teneurin-4 promotes homophilic and heterophilic cell binding with all teneurins.•Oligodendrocyte lineage cells adhere to the extracellular domain of teneurins.•The homophilic interaction of teneurin-4 promotes oligodendrocyte differentiation.
The gap junctions (GJs) formed by Cx40 and Cx43 provide a low resistance passage allowing for rapid propagation of action potentials. Sporadic somatic mutations in GJA5 (encoding Cx40) have been ...identified in lone atrial fibrillation (AF) patients. More recently germline autosomal dominantly inherited mutations in GJA5 have been found in early onset lone AF patients in several families over generations. Characterizations of these AF-linked Cx40 mutants in model cells and in patient tissues revealed that some of the mutants reduced GJ channel function due to an impaired trafficking or channel formation. While others showed a gain-of-function in hemichannels. These functional alterations in GJs or hemichannel may play an important role in the pathogenesis of AF in the mutant carriers.
•Successful expression of Halo-tagged ECD of CaSR in E. coli cell.•Rapid preparation of the immobilized CaSR column without the protein purification.•Revealing the interaction mechanism of three ...aminoglycoside agonists with CaSR.•Recognizing novel ligands targeting CaSR from Chinese herb medicine ‘Shuangdan’.
Human calcium-sensing receptor (CaSR), a member of the G-protein-coupled receptor superfamily (GPCR), has been a therapeutic target for developing new drugs against calciotropic disorders and non-calciotropic diseases. The highly efficient methodologies for pursuing novel ligands/drugs remained a challenge due to the redundant purification processes of membrane protein in some widely-used methods including NMR, X-ray crystallography, Fluorescence Titration Spectroscopy, and Circular Dichroism. Herein, extracellular domain (ECD) of CaSR as its functional fragment was used to develop a rapid chromatographic method, which involved the synthesis of stationary phase material based on the site-specific covalent reaction of Halogenated alkane dehalogenase (Halo)-tagged ECD of CaSR in cell lysate with 6-chlorocaproic acid modified silica beads, the use of the immobilized CaSR column for revealing the interaction of three known agonists with CaSR and further screening ligands from complex matrix like Chinese herb medicine ‘Shuangdan’. The immobilized CaSR column was prepared rapidly without the protein purification and retained a good stability and specificity for at least 35 days. It was revealed that one type of binding sites occurred on CaSR with the binding affinity of neomycin > gentamicin-C / kanamycin, presumably which related to the number of structural amino groups attached. This method allowed for recognizing specifically novel ligands from ’Shuangdan’, demonstrating one type of binding sites on CaSR with the binding affinity of gallic acid > caffeic acid > paeonol. These results indicated that, the immobilization of a representative extracellular domain of CaSR to silica beads as biomaterial is feasible to develop a new rapid method, which can be successfully applied in screening novel ligands efficiently from complex matrices.
Non-small cell lung cancer (NSCLC) is one of the most general malignant tumors. The overexpression of epidermal growth factor receptor (EGFR) is a common marker in NSCLC, and it plays an important ...role in the proliferation, invasion, and metastasis of cancer cells. At present, drugs developed with EGFR as a target suffer from drug resistance, so it is necessary to study new compounds for the treatment of NSCLC. The active substance in green tea is EGCG, which has anti-cancer effects. In this study, we synthesized dimeric-(−)-epigallocatechin-3-gallate (prodelphinidin B-4-3,3‴-di-O-gallate, PBOG), and explored the effect of PBOG on lung cancer cells. PBOG can inhibit the proliferation and migration of NCI–H1975 cells, promote cell apoptosis, and inhibit cell cycle progression. In addition, PBOG can bind to the EGFR ectodomain protein and change the secondary structure of the protein. At the same time, PBOG decreases the expression of EGFR and downstream protein phosphorylation. Animal experiments confirmed that PBOG can inhibit tumor growth by inhibiting EGFR phosphorylation. Collectively, our study results show that PBOG may induce a decrease in intracellular phosphorylated EGFR expression by binding to the EGFR ectodomain protein, thereby inducing apoptosis and inhibiting cell cycle progression, thus providing a new strategy to treat lung cancer.
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•PBOG can inhibit cell proliferation and cell cycle processes，and promote apoptosis.•PBOG may combine with EGFR extracellular domain to change its secondary structure.•PBOG inhibits the phosphorylation of EGFR in NCI–H1975 cells.•PBOG can inhibit tumor growth in vivo by reducing p-EGFR protein.
Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface ...displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass‐sensing RsgI‐type anti‐σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti‐σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn‐Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti‐σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.
Receptor kinases (RK) constitute the largest protein kinase family in plants. In particular, members of the leucine-rich repeat-receptor kinases (LRR-RKs) are involved in the perception of various ...signals at the plasma membrane. Experimental evidence over the past years revealed a conserved activation mechanism through ligand-inducible heterodimer formation: a ligand is recognized by a receptor kinase with a large extracellular domain (ECD). This ligand binding receptor directly interacts with a so-called co-receptor with a small ECD for ligand fixation and kinase activation. A large proportion of LRR-RKs is functionally still uncharacterized and the dynamic complexity of the plasma membrane makes it difficult to precisely define receptor kinase heterodimer pairs and their functions. In this review, we give an overview of the current knowledge of LRR receptor and co-receptor functions. We use ECD lengths to classify the LRR receptor kinase family and describe different interaction properties of ligand-binding receptors and their respective co-receptor from a network perspective.
The parathyroid hormone type 1 receptor (PTHR1), a Class B GPCR, is activated by long polypeptides, including drugs for osteoporosis and hypoparathyroidism. The PTHR1 engages peptide agonists via a ...two-step mechanism. Initial contact involves the extracellular domain (ECD), which has been thought to contribute primarily to receptor-peptide binding, and then the N terminus of the peptide engages the receptor transmembrane domain (TMD), which is thought to control the message conveyed to intracellular partners. This mechanism has been suggested to apply to other Class B GPCRs as well. Here, we show that modification of a PTHR1 agonist at ECD-contact sites can alter the signaling profile, an outcome that is not accommodated by the current two-step binding model. Our data support a modified two-step binding model in which agonist orientation on the ECD surface can influence the geometry of agonist-TMD engagement. This expanded binding model offers a mechanism by which altering ECD-contact residues can affect signaling profile. Our discoveries provide a rationale for exploring agonist modifications distal from the TMD-contact region in future efforts to optimize therapeutic performance of peptide hormone analogs.
Dimerization of the CNNM extracellular domain Shahsavan, Ashkan; Lee, Emma L.; Illes, Katalin ...
Protein science,
February 2024, 2024-Feb, 2024-02-00, 20240201, Volume:
33, Issue:
2
Journal Article
Peer reviewed
Open access
Cystathionine‐β$$ \beta $$‐synthase (CBS)‐pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They mediate magnesium ...homeostasis directly by transport of Mg2+ ions and indirectly by regulation of the transient receptor potential ion channel subfamily M member 7 (TRPM7). Here, we report the crystal structure of the extracellular domain of tapeworm CNNM4. The domain forms a dimer of immunoglobulin‐like (Ig‐like) folds with electron density observed for three glycosylation sites. Analytical ultracentrifugation confirms that mutations in the extracellular domain of human CNNM4 prevent its dimerization. An analogous mutation in mouse CNNM2 impairs its activity in a cellular assay of Mg2+ transport.
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