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1. Development of an antibody fragment that stabilizes GPCR/G-protein complexes.

Author

Maeda, Shoji, Koehl, Antoine, Matile, Hugues, Hu, Hongli, Hilger, Daniel, Schertler, Gebhard FX, Manglik, Aashish, Skiniotis, Georgios, Dawson, Roger JP, and Kobilka, Brian K

Subjects
Humans, Macromolecular Substances, GTP-Binding Proteins, Nucleotides, Rhodopsin, Immunoglobulin Fragments, Receptors, G-Protein-Coupled, Antibodies, Monoclonal, Cryoelectron Microscopy, Crystallography, X-Ray, Protein Engineering, Signal Transduction, Binding Sites, Protein Structure, Secondary, Protein Binding, Protein Domains, Antibodies, Monoclonal, Crystallography, X-Ray, Protein Structure, Secondary, Receptors, G-Protein-Coupled
Abstract

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.

Published
2018

2. Structure of the µ-opioid receptor-Gi protein complex.

Author

Koehl, Antoine, Hu, Hongli, Maeda, Shoji, Zhang, Yan, Qu, Qianhui, Paggi, Joseph M, Latorraca, Naomi R, Hilger, Daniel, Dawson, Roger, Matile, Hugues, Schertler, Gebhard FX, Granier, Sebastien, Weis, William I, Dror, Ron O, Manglik, Aashish, Skiniotis, Georgios, and Kobilka, Brian K

Subjects
Animals, Mice, Inbred BALB C, Humans, Mice, Morphinans, GTP-Binding Protein alpha Subunits, Gi-Go, GTP-Binding Protein alpha Subunits, Gs, Enkephalin, Ala(2)-MePhe(4)-Gly(5)-, Receptors, Adrenergic, beta-2, Receptors, Opioid, mu, Ligands, Cryoelectron Microscopy, Binding Sites, Substrate Specificity, Female, Protein Stability, Molecular Dynamics Simulation, Inbred BALB C, GTP-Binding Protein alpha Subunits, Gi-Go, Gs, Enkephalin, Ala(2)-MePhe(4)-Gly(5)-, Receptors, Adrenergic, beta-2, Opioid, mu, General Science & Technology
Abstract

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.

Published
2018

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