Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor (#211)

Brian P Cary 1 2 3 , Giuseppe Deganutti 4 , Peishen Zhao 2 3 , Tin T Truong 3 , Sarah J Piper 2 3 , Xinyu Liu 1 , Matthew J Belousoff 2 3 , Danev Radostin 5 , Patrick M Sexton 2 3 , Denise Wootten 2 3 , Samuel H Gellman 1
  1. Department of Chemistry, University of Wisconsin-Madison, Madison, WI, United States
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Parkville, Vic, Australia
  3. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
  4. Centre for Sport, Exercise, and Life Sciences, Faculty of Health and Life Sciences, Coventry University, Coventry, UK
  5. Graduate School of Medicine, University of Tokyo, Tokyo, Japan

Recent advances in structural biology have enabled determination of structures of all 15 class B1 G protein-coupled receptors (GPCRs). While these snapshots provide insights into receptor activation mechanisms, the importance of bound agonist dynamics is underappreciated. Here, we directly observe conformational plasticity in the N-terminus of  a class B1 GPCR agonist and provide evidence that these dynamics are vital for activity.1

The glucagon-like peptide 1 receptor (GLP-1R) is an archetypal class B1 GPCR, a critical regulator of glucose homeostasis, and an established target for the treatment of type II diabetes and obesity.2 In recent, full-length structures of the GLP-1R, the critical N-terminus of GLP-1 is rigidly bound in a helical state.3 However, previous studies postulated the existence of a helix-capping motif in class B1 hormones in this region.4 To better understand the determinants of GLP-1R activation by peptide hormones, we systematically probed the N-terminus of GLP-1 and exenatide, a high affinity, high potency, GLP-1R agonist drug, by incorporating single amino acid substitutions in place of a highly conserved glycine residue. We found that disfavoring agonist helicity by incorporation of a single D-alanine residue minimally affects potency but decreases affinity. Conversely, incorporation of another unnatural amino acid, one that readily adopts a helix, abrogates activity but not affinity, yielding a potent inhibitor. These data suggest that the ability of GLP-1 to access non-helical conformations is important for activity. We then used single particle cryo-electron microscopy (cryo-EM) to visualize a potent agonist containing D-alanine bound to GLP-1R/Gs heterotrimer. 3D classification and 3D variance analysis of the cryo-EM data revealed that, unlike GLP-1, the N-terminal region of this analogue transited through multiple conformations while bound to the receptor. Molecular dynamics simulations supported the hypothesis of a mobile, unwinding peptide N-terminus concurrent with outward extracellular loop 3 (ECL3) movement that was also observed in analysis of the cryo-EM data. Collectively, our evidence supports the hypothesis that agonist mobility is important for the activation of the GLP-1R.

  1. 1 Cary, B. P. et al. Structural and functional diversity among agonist-bound states of the GLP-1 receptor. Nat Chem Biol 18, 256–263 (2022).
  2. 2. Graaf, C. d. et al. Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes. Pharmacological Reviews 68, 954–1013 (2016).
  3. 3. Zhang, X. et al. Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists. Molecular Cell 80, 485-500.e7 (2020).
  4. 4. Neumann, J.-M. et al. Class-B GPCR activation: is ligand helix-capping the key? Trends in Biochemical Sciences 33, 314–319 (2008).