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Author(s): Hai-Qiang Dai [sup.1] [sup.2] , Hongli Hu [sup.1] [sup.2] , Jiangman Lou [sup.1] [sup.2] , Adam Yongxin Ye [sup.1] [sup.2] , Zhaoqing Ba [sup.1] [sup.2] , Xuefei Zhang [sup.1] [...], RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a J.sub.H-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs.sup.1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning.sup.3. Scanning of RAG from the DJ.sub.H-RC-RSS to upstream convergent V.sub.H-RSSs is impeded by D-proximal CTCF-binding elements (CBEs).sup.2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the V.sub.H locus that is proposed to provide distal V.sub.Hs access to the DJ.sub.H-RC.sup.6-9. Here we report that an inversion of the entire 2.4-Mb V.sub.H locus in mouse primary progenitor B cells abrogates rearrangement of both V.sub.H-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic V.sub.H-RSSs that are normally in opposite orientation and RAG scanning beyond the V.sub.H locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the V.sub.H locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL).sup.10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the V.sub.H locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism. Long-distance V(D)J recombination is facilitated by contraction of the Igh locus and linear RAG scanning along chromatin, both driven by cohesin-mediated loop extrusion, which allows recombination of widely separated gene segments to occur.

Author(s): Dean P. Staus [sup.1] [sup.2] , Hongli Hu [sup.3] [sup.8] , Michael J. Robertson [sup.3] [sup.4] , Alissa L. W. Kleinhenz [sup.1] [sup.2] [sup.9] , Laura M. Wingler [sup.1] [...], After activation by an agonist, G-protein-coupled receptors (GPCRs) recruit [beta]-arrestin, which desensitizes heterotrimeric G-protein signalling and promotes receptor endocytosis.sup.1. Additionally, [beta]-arrestin directly regulates many cell signalling pathways that can induce cellular responses distinct from that of G proteins.sup.2. In contrast to G proteins, for which there are many high-resolution structures in complex with GPCRs, the molecular mechanisms underlying the interaction of [beta]-arrestin with GPCRs are much less understood. Here we present a cryo-electron microscopy structure of [beta]-arrestin 1 ([beta]arr1) in complex with M2 muscarinic receptor (M2R) reconstituted in lipid nanodiscs. The M2R-[beta]arr1 complex displays a multimodal network of flexible interactions, including binding of the N domain of [beta]arr1 to phosphorylated receptor residues and insertion of the finger loop of [beta]arr1 into the M2R seven-transmembrane bundle, which adopts a conformation similar to that in the M2R-heterotrimeric G.sub.o protein complex.sup.3. Moreover, the cryo-electron microscopy map reveals that the C-edge of [beta]arr1 engages the lipid bilayer. Through atomistic simulations and biophysical, biochemical and cellular assays, we show that the C-edge is critical for stable complex formation, [beta]arr1 recruitment, receptor internalization, and desensitization of G-protein activation. Taken together, these data suggest that the cooperative interactions of [beta]-arrestin with both the receptor and the phospholipid bilayer contribute to its functional versatility. A cryo-electron microscopy structure of [beta]-arrestin 1 in complex with the M2 muscarinic receptor reconstituted in lipid nanodiscs is reported.

Author(s): Yu Zhang [sup.1] [sup.2] [sup.3] [sup.4] [sup.13] , Xuefei Zhang [sup.1] [sup.2] [sup.3] , Zhaoqing Ba [sup.1] [sup.2] [sup.3] , Zhuoyi Liang [sup.1] [sup.2] [sup.3] , Edward W. Dring [...], The RAG endonuclease initiates Igh V(D)J assembly in B cell progenitors by joining D segments to J.sub.H segments, before joining upstream V.sub.H segments to DJ.sub.H intermediates.sup.1. In mouse progenitor B cells, the CTCF-binding element (CBE)-anchored chromatin loop domain.sup.2 at the 3' end of Igh contains an internal subdomain that spans the 5' CBE anchor (IGCR1).sup.3, the D.sub.H segments, and a RAG-bound recombination centre (RC).sup.4. The RC comprises the J.sub.H-proximal D segment (DQ52), four J.sub.H segments, and the intronic enhancer (iE[mu]).sup.5. Robust RAG-mediated cleavage is restricted to paired V(D)J segments flanked by complementary recombination signal sequences (12RSS and 23RSS).sup.6. D segments are flanked downstream and upstream by 12RSSs that mediate deletional joining with convergently oriented J.sub.H-23RSSs and V.sub.H-23RSSs, respectively.sup.6. Despite 12/23 compatibility, inversional D-to-J.sub.H joining via upstream D-12RSSs is rare.sup.7,8. Plasmid-based assays have attributed the lack of inversional D-to-J.sub.H joining to sequence-based preference for downstream D-12RSSs.sup.9, as opposed to putative linear scanning mechanisms.sup.10,11. As RAG linearly scans convergent CBE-anchored chromatin loops.sup.4,12-14, potentially formed by cohesin-mediated loop extrusion.sup.15-18, we revisited its scanning role. Here we show that the chromosomal orientation of J.sub.H-23RSS programs RC-bound RAG to linearly scan upstream chromatin in the 3' Igh subdomain for convergently oriented D-12RSSs and, thereby, to mediate deletional joining of all D segments except RC-based DQ52, which joins by a diffusion-related mechanism. In a DQ52-based RC, formed in the absence of J.sub.H segments, RAG bound by the downstream DQ52-RSS scans the downstream constant region exon-containing 3' Igh subdomain, in which scanning can be impeded by targeted binding of nuclease-dead Cas9, by transcription through repetitive Igh switch sequences, and by the 3' Igh CBE-based loop anchor. Each scanning impediment focally increases RAG activity on potential substrate sequences within the impeded region. High-resolution mapping of chromatin interactions in the RC reveals that such focal RAG targeting is associated with corresponding impediments to the loop extrusion process that drives chromatin past RC-bound RAG. V(D)J recombination in B cells involves cohesin-mediated extrusion of chromatin loops to present DNA targets for cleavage and joining.

Author(s): Hideaki E. Kato [sup.1] [sup.2] , Yan Zhang [sup.1] [sup.3] [sup.9] , Hongli Hu [sup.1] [sup.3] , Carl-Mikael Suomivuori [sup.1] [sup.3] [sup.4] [sup.5] , Francois Marie Ngako Kadji [sup.6] [...], Neurotensin receptor 1 (NTSR1) is a G-protein-coupled receptor (GPCR) that engages multiple subtypes of G protein, and is involved in the regulation of blood pressure, body temperature, weight and the response to pain. Here we present structures of human NTSR1 in complex with the agonist JMV449 and the heterotrimeric G.sub.i1 protein, at a resolution of 3 Å. We identify two conformations: a canonical-state complex that is similar to recently reported GPCR-G.sub.i/o complexes (in which the nucleotide-binding pocket adopts more flexible conformations that may facilitate nucleotide exchange), and a non-canonical state in which the G protein is rotated by about 45 degrees relative to the receptor and exhibits a more rigid nucleotide-binding pocket. In the non-canonical state, NTSR1 exhibits features of both active and inactive conformations, which suggests that the structure may represent an intermediate form along the activation pathway of G proteins. This structural information, complemented by molecular dynamics simulations and functional studies, provides insights into the complex process of G-protein activation. Cryo-electron microscopy structures of human neurotensin receptor 1 in complex with G.sub.i1 protein and the agonist JMV449 reveal a non-canonical state that may represent an intermediate form in G-protein activation.

Author(s): Antoine Koehl [sup.1] , Hongli Hu [sup.1] [sup.2] , Dan Feng [sup.3] , Bingfa Sun [sup.3] , Yan Zhang [sup.1] [sup.2] , Michael J. Robertson [sup.1] [sup.2] , Matthew [...], Metabotropic glutamate receptors are family C G-protein-coupled receptors. They form obligate dimers and possess extracellular ligand-binding Venus flytrap domains, which are linked by cysteine-rich domains to their 7-transmembrane domains. Spectroscopic studies show that signalling is a dynamic process, in which large-scale conformational changes underlie the transmission of signals from the extracellular Venus flytraps to the G protein-coupling domains--the 7-transmembrane domains--in the membrane. Here, using a combination of X-ray crystallography, cryo-electron microscopy and signalling studies, we present a structural framework for the activation mechanism of metabotropic glutamate receptor subtype 5. Our results show that agonist binding at the Venus flytraps leads to a compaction of the intersubunit dimer interface, thereby bringing the cysteine-rich domains into close proximity. Interactions between the cysteine-rich domains and the second extracellular loops of the receptor enable the rigid-body repositioning of the 7-transmembrane domains, which come into contact with each other to initiate signalling.The activation mechanism of metabotropic glutamate receptor subtype 5, a member of the family C G-protein-coupled receptors, is characterized by a combination of cryo-electron microscopy, crystallography and signalling studies.

Author(s): Antoine Koehl [sup.1] , Hongli Hu [sup.1] [sup.2] , Shoji Maeda [sup.2] , Yan Zhang [sup.1] [sup.2] , Qianhui Qu [sup.1] [sup.2] , Joseph M. Paggi [sup.1] [sup.2] [sup.3] [...], The [mu]-opioid receptor ([mu]OR) 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 [mu]OR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein G.sub.i. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the [mu]OR bound to the agonist peptide DAMGO and nucleotide-free G.sub.i. 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 [mu]OR-G.sub.i complex to previously determined structures of other GPCRs bound to the stimulatory G protein G.sub.s reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein [alpha]-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the G.sub.i protein-coupling specificity of the [micro]OR. A cryo-electron structure of the [micro]-opioid receptor in complex with the peptide agonist DAMGO and the inhibitory G protein G.sub.i reveals structural determinants of its G protein-binding specificity.

Author(s): Yan Zhang [1]; Bingfa Sun [2]; Dan Feng [2]; Hongli Hu [1]; Matthew Chu [2]; Qianhui Qu [1]; Jeffrey T. Tarrasch [1]; Shane Li [2]; Tong Sun Kobilka [2]; [...], Glucagon-like peptide 1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to the GLP-1 receptor (GLP-1R), a class B G-protein-coupled receptor (GPCR) that signals primarily through the stimulatory G protein G[sub.s]. Class B GPCRs are important therapeutic targets; however, our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we report the cryo-electron microscopy structure of the peptide-activated GLP-1RG[sub.s] complex at near atomic resolution. The peptide is clasped between the N-terminal domain and the transmembrane core of the receptor, and further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the 5-helix of the Ras-like domain of G[sub.s]. These results provide a structural framework for understanding class B GPCR activation through hormone binding.

Author(s): Antoine Koehl [sup.1] , Hongli Hu [sup.1] [sup.2] , Dan Feng [sup.3] , Bingfa Sun [sup.3] , Yan Zhang [sup.1] [sup.2] , Michael J. Robertson [sup.1] [sup.2] , Matthew [...], The surname of author Toon Laeremans was misspelled 'Laermans'. This error has been corrected online.