Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT 1B G protein–coupled ...receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT 2B receptor, the 5-HT 1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.
Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class ...B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family.
The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the ...3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen-deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.
Apelin receptor (APJR) is a key regulator of human cardiovascular function and is activated by two different endogenous peptide ligands, apelin and Elabela, each with different isoforms diversified ...by length and amino acid sequence. Here we report the 2.6-Å resolution crystal structure of human APJR in complex with a designed 17-amino-acid apelin mimetic peptide agonist. The structure reveals that the peptide agonist adopts a lactam constrained curved two-site ligand binding mode. Combined with mutation analysis and molecular dynamics simulations with apelin-13 binding to the wild-type APJR, this structure provides a mechanistic understanding of apelin recognition and binding specificity. Comparison of this structure with that of other peptide receptors suggests that endogenous peptide ligands with a high degree of conformational flexibility may bind and modulate the receptors via a similar two-site binding mechanism.
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•Designed peptide mimic AMG3054 with reduced conformational flexibility•Structure of apelin receptor in complex with AMG3054•Designed peptide AMG3054 adopts a lactam constrained curved structure•A two-site binding mode is observed for peptide GPCR recognition
Ma et al. present an atomic-resolution structure of the human apelin receptor (APJR, a class A G-protein-coupled receptor) in complex with a 17-residue apelin peptide mimic by X-ray crystallography. This structure reveals a two-site ligand binding mode that has not been seen in any other solved class A receptor structures.
Cysteinyl leukotriene G protein-coupled receptors CysLT
and CysLT
regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT
R has been used for ...treating asthma and associated diseases for over two decades, CysLT
R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT
R in complex with three dual CysLT
R/CysLT
R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants.
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the ...crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.
Platelet-activating-factor receptor (PAFR) responds to platelet-activating factor (PAF), a phospholipid mediator of cell-to-cell communication that exhibits diverse physiological effects. PAFR is ...considered an important drug target for treating asthma, inflammation and cardiovascular diseases. Here we report crystal structures of human PAFR in complex with the antagonist SR 27417 and the inverse agonist ABT-491 at 2.8-Å and 2.9-Å resolution, respectively. The structures, supported by molecular docking of PAF, provide insights into the signal-recognition mechanisms of PAFR. The PAFR-SR 27417 structure reveals an unusual conformation showing that the intracellular tips of helices II and IV shift outward by 13 Å and 4 Å, respectively, and helix VIII adopts an inward conformation. The PAFR structures, combined with single-molecule FRET and cell-based functional assays, suggest that the conformational change in the helical bundle is ligand dependent and plays a critical role in PAFR activation, thus greatly extending knowledge about signaling by G-protein-coupled receptors.
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms
. As central mediators in this complex ...signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.
A flux pump is a kind of power source that energizes superconducting magnets usually embedded in the cryogenic system. Therefore, heat loss through current leads is eliminated, allowing for more ...efficient cooling design. A winding technique, the so-called "wind-and-flip," had been proposed by the authors to realize a jointless HTS coil for a perfect resistanceless closed loop, which may enable to induce a persistent current in an HTS coil wound with second-generation HTS tape. In this paper, we investigated charging/discharging characteristics and a possibility of persistent current operation of a jointless HTS coil with a flux pump. Results are discussed with regard to current capacity and temporal stability with a prototype jointless HTS coil.
With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have ...an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p -acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing k cₐₜ, but not significantly affecting K M. To understand the structural basis for this enhanced activity, we solved the X-ray crystal structures of the ligand-free mutant enzyme and of the deacylation-defective wild-type and mutant cephalexin acyl-enzyme intermediates. These structures show that the Val-216–AcrF mutation leads to conformational changes in key active site residues—both in the free enzyme and upon formation of the acyl-enzyme intermediate—that lower the free energy of activation of the substrate transacylation reaction. The functional changes induced by this mutation could not be reproduced by substitution of any of the 20 canonical amino acids for Val-216, indicating that an expanded genetic code may offer novel solutions to proteins as they evolve new activities.
Significance We describe a general strategy that begins to allow us to address the question of whether an expanded genetic code provides an evolutionary advantage to an organism. A large library of β-lactamase variants with distinct noncanonical amino acids substituted randomly at single sites throughout the protein was generated and then subjected to an antibiotic growth-based screen to identify mutants with enhanced catalytic activity. We show that a unique noncanonical mutation in the enzyme β-lactamase significantly increases catalytic activity by unexpected mechanisms. These effects cannot be recapitulated by other canonical amino acids at this site, suggesting that an expanded set of building blocks beyond the canonical 20 may offer unique solutions to organisms in the evolution of new functions.
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