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The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and β-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance β-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.Here, using cryo-electron microscopy and solution NMR, the authors reveal the structural and dynamic mechanisms underlying the signaling versatility of a muscarinic receptor regulated by orthosteric and allosteric ligands. [ABSTRACT FROM AUTHOR]

Heterotrimeric G proteins are categorized into four main families based on their function and sequence, Gs, Gi/o, Gq/11, and G12/13. One receptor can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. However, the precise mechanism underlying different coupling efficiencies is unknown. Here, we study the structural mechanism underlying primary and secondary Gi/o coupling, using the muscarinic acetylcholine receptor type 2 (M2R) as the primary Gi/o-coupling receptor and the β2-adrenergic receptor (β2AR, which primarily couples to Gs) as the secondary Gi/o-coupling receptor. Hydrogen/deuterium exchange mass spectrometry and mutagenesis studies reveal that the engagement of the distal C-terminus of Gαi/o with the receptor differentiates primary and secondary Gi/o couplings. This study suggests that the conserved hydrophobic residue within the intracellular loop 2 of the receptor (residue 34.51) is not critical for primary Gi/o-coupling; however, it might be important for secondary Gi/o-coupling. G protein-coupled receptors (GPCRs) can couple to more than one G protein subtype, and the coupling efficiency varies depending on the GPCR-G protein pair. Here authors use hydrogen/deuterium exchange mass spectrometry and mutagenesis to study the structural mechanism underlying primary and secondary Gi/o coupling. [ABSTRACT FROM AUTHOR]

The μ-opioid receptor (μOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by μOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the μOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the μOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR. A cryo-electron structure of the µ-opioid receptor in complex with the peptide agonist DAMGO and the inhibitory G protein Gi reveals structural determinants of its G protein-binding specificity. [ABSTRACT FROM AUTHOR]

We present comprehensive maps at single-amino acid resolution of the residues stabilizing the human Gαi1 subunit in nucleotide- and receptor-bound states. We generated these maps by measuring the effects of alanine mutations on the stability of Gαi1 and the rhodopsin-Gαi1 complex. We identified stabilization clusters in the GTPase and helical domains responsible for structural integrity and the conformational changes associated with activation. In activation cluster I, helices α1 and α5 pack against strands β1-β3 to stabilize the nucleotide-bound states. In the receptor-bound state, these interactions are replaced by interactions between α5 and strands β4-β6. Key residues in this cluster are Y320, which is crucial for the stabilization of the receptor-bound state, and F336, which stabilizes nucleotide-bound states. Destabilization of helix α1, caused by rearrangement of this activation cluster, leads to the weakening of the interdomain interface and release of GDP. [ABSTRACT FROM AUTHOR]

Gap junctions consist of arrays of intercellular channels composed of integral membrane proteins called connexin in vertebrates. Gap junction channels regulate the passage of ions and biological molecules between adjacent cells and, therefore, are critically important in many biological activities, including development, differentiation, neural activity, and immune response. Mutations in connexin genes are associated with several human diseases, such as neurodegenerative disease, skin disease, deafness, and developmental abnormalities. The activity of gap junction channels is regulated by the membrane voltage, intracellular microenvironment, interaction with other proteins, and phosphorylation. Each connexin channel has its own property for conductance and molecular permeability. A number of studies have tried to reveal the molecular architecture of the channel pore that should confer the connexin-specific permeability/selectivity properties and molecular basis for the gating and regulation. In this review, we give an overview of structural studies and describe the structural and functional relationship of gap junction channels. [ABSTRACT FROM AUTHOR]

Gap junctions consist of arrays of intercellular channels between adjacent cells that permit the exchange of ions and small molecules. Here we report the crystal structure of the gap junction channel formed by human connexin 26 (Cx26, also known as GJB2) at 3.5 Å resolution, and discuss structural determinants of solute transport through the channel. The density map showed the two membrane-spanning hemichannels and the arrangement of the four transmembrane helices of the six protomers forming each hemichannel. The hemichannels feature a positively charged cytoplasmic entrance, a funnel, a negatively charged transmembrane pathway, and an extracellular cavity. The pore is narrowed at the funnel, which is formed by the six amino-terminal helices lining the wall of the channel, which thus determines the molecular size restriction at the channel entrance. The structure of the Cx26 gap junction channel also has implications for the gating of the channel by the transjunctional voltage. [ABSTRACT FROM AUTHOR]

Ten cases with iliac artery disease were evaluated using rotational angiography with revolution of the arm of the X-ray tube support unit. The tip of the catheter was advanced to the infrarenal aorta, and contrast medium was injected at a rate of 6–10 ml/second. Digital angiographic images from multiple projection angles were acquired at 15 frames/second with a rotation speed at about 10°/second and a range of greater than 90°. This method made it possible to detect ulcerations of the artery, which could not be visualized using conventional single-plane angiography. The three-dimensional structure of a dissection could also be visualized. In seven cases of occlusive iliac artery disease, comparison with rotational angiography revealed that anteroposterior views of single-plane angiography underestimated the extent of the disease in four cases and overestimated the extent of the disease in one case. In two cases, no additional clinical information was obtained. In conclusion, rotational angiography provides accurate three-dimensional information concerning the lumen of the iliac arteries. [ABSTRACT FROM AUTHOR]

We treated a Japanese man with aneurysmal dilatation of the bovine graft transplanted for femorofemoral bypass. The reconstruction was performed successfully by the replacement with a Dacron graft 29 months after the first operation. Aneurysmal dilatation was observed over the entire length of the graft, and the wall of the graft was fragile. Disruption of collagen fibers and bleeding into the graft wall from the arteries which had been newly formed in abundance were extensive. The histological findings suggested that bovine grafts should be used for only a short period or for the replacement of small arteries. [ABSTRACT FROM AUTHOR]

Single-particle cryo-electron microscopy (cryo-EM) has recently enabled high-resolution structure determination of numerous biological macromolecular complexes. Despite this progress, the application of high-resolution cryo-EM to G protein coupled receptors (GPCRs) in complex with heterotrimeric G proteins remains challenging, owning to both the relative small size and the limited stability of these assemblies. Here we describe the development of antibody fragments that bind and stabilize GPCR-G protein complexes for the application of high-resolution cryo-EM. One antibody in particular, mAb16, stabilizes GPCR/G-protein complexes by recognizing an interface between Gα and Gβγ subunits in the heterotrimer, and confers resistance to GTPγS-triggered dissociation. The unique recognition mode of this antibody makes it possible to transfer its binding and stabilizing effect to other G-protein subtypes through minimal protein engineering. This antibody fragment is thus a broadly applicable tool for structural studies of GPCR/G-protein complexes. [ABSTRACT FROM AUTHOR]