The class B glucagon-like peptide-1 (GLP-1) G protein-coupled receptor is a major target for the treatment of type 2 diabetes and obesity. Endogenous and mimetic GLP-1 peptides exhibit biased agonism-a difference in functional selectivity-that may provide improved therapeutic outcomes. Here we describe the structure of the human GLP-1 receptor in complex with the G protein-biased peptide exendin-P5 and a Gα heterotrimer, determined at a global resolution of 3.3 Å. At the extracellular surface, the organization of extracellular loop 3 and proximal transmembrane segments differs between our exendin-P5-bound structure and previous GLP-1-bound GLP-1 receptor structure. At the intracellular face, there was a six-degree difference in the angle of the Gαs-α5 helix engagement between structures, which was propagated across the G protein heterotrimer. In addition, the structures differed in the rate and extent of conformational reorganization of the Gα protein. Our structure provides insights into the molecular basis of biased agonism.
Peptide drugs targeting class B1 GPCRs can treat multiple diseases, however there remains substantial interest in the development of orally delivered non-peptide drugs. Here we reveal unexpected overlap between signalling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist, PF 06882961, and GLP-1 that was not observed for another compound, OWL-833. Both compounds are currently in clinical trials for treatment of type 2 diabetes. High resolution cryo-EM structures reveal the binding sites for PF-06882961 and GLP-1 substantially overlap, whereas OWL-833 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not OWL-833, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of nonpeptide agonists targeting class B GPCRs.
Highlights d Cryo-EM structure reveals how CRF1R interacts with CRF and the Gs signaling protein d Cryo-EM structure reveals interactions of Pac1nR with PACAP-38 and Gs d Evolutionary related GPCRs have greater conservation in peptide and G protein binding
Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:Gαi2βγ and calcitonin gene-related peptide (CGRP):CLR:RAMP1:Gαsβγ:Nb35 were purified to homogeneity and were stable in negative stain electron microscopy. These were suitable for structure determination by cryo-electron microscopy at 3.6 and 3.3 Å resolution, respectively. The dominant negative Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.
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