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Structural and functional diversity among agonist-bound states of the GLP-1 receptor

Abstract

Recent advances in G-protein-coupled receptor (GPCR) structural elucidation have strengthened previous hypotheses that multidimensional signal propagation mediated by these receptors depends, in part, on their conformational mobility; however, the relationship between receptor function and static structures is inherently uncertain. Here, we examine the contribution of peptide agonist conformational plasticity to activation of the glucagon-like peptide 1 receptor (GLP-1R), an important clinical target. We use variants of the peptides GLP-1 and exendin-4 (Ex4) to explore the interplay between helical propensity near the agonist N terminus and the ability to bind to and activate the receptor. Cryo-EM analysis of a complex involving an Ex4 analog, the GLP-1R and Gs heterotrimer revealed two receptor conformers with distinct modes of peptide–receptor engagement. Our functional and structural data, along with molecular dynamics (MD) simulations, suggest that receptor conformational dynamics associated with flexibility of the peptide N-terminal activation domain may be a key determinant of agonist efficacy.

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Fig. 1: Probing the relationship between agonist N-terminal conformational propensity and receptor activation with single substitutions.
Fig. 2: Diverse measures of GLP-1R engagement.
Fig. 3: Cryo-EM structure of Ex4-d-Ala bound to the GLP-1R in complex with the heterotrimeric G protein and nanobody 35.
Fig. 4: Comparisons of conformers 1 and 2 for Ex4-d-Ala bound to GLP-1R.
Fig. 5: MD simulations of Ex4-d-Ala bound to GLP-1R and a proposed, simplified energy landscape for the interaction of GLP-1R with peptide agonists.

Data availability

Sequencing data for NLuc-GLP-1R is available at Addgene (ID 124831). Atomic coordinates and cryo-EM density maps for Ex4-d-Ala-bound GLP-1R–Gs in conformer 1 and conformer 2 have been deposited in the PDB under accession numbers 7S1M and 7S3I and Electron Microscopy Data Bank entries EMD-24805 and EMD-24825, respectively. MD trajectories are available at https://doi.org/10.5281/zenodo.5578864. Source data are provided with this paper.

Code availability

No new code was used in this study. A list of software used is available in the Methods section and Reporting Summary.

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Acknowledgements

This work was supported by the National Institutes of Health (R01 GM056414, to S.H.G.). B.P.C. was supported in part by a graduate fellowship from the NSF (DGE-1747503) and by a Biotechnology Training Grant from NIGMS (T32 GM008349). R.D. was supported by a Takeda Science Foundation 2019 Medical Research Grant and Japan Science and Technology Agency PRESTO (18069571). P.M.S. and D.W. were supported by an ARC Centre Grant (IC200100052). P.M.S. was supported by the National Health and Medical Research Council of Australia (NHMRC) Program Grant (1150083) and Senior Principal Research Fellowship (1154434). D.W. was supported by NHMRC Project Grants (1126857 and 1184726) and a NHMRC Senior Research Fellowship (1155302). This study made use of the National Magnetic Resonance Facility at Madison, which is supported by NIH grants P41GM136463 and P41RR002301; equipment was purchased with funds from the University of Wisconsin-Madison, the NIH (P41GM103399, S10RR02781, S10RR08438, S10RR023438, S10RR025062, S10RR029220) and the NSF (DMB-8415048, OIA-9977486, BIR-9214394). We thank Promega (Madison, WI) for sharing plasmid DNA encoding NLuc.

Author information

Authors and Affiliations

Authors

Contributions

B.P.C. designed the project, synthesized peptides, generated the NLuc-GLP-1R construct and expressed and purified the protein complex. G.D. performed and analyzed MD simulations. B.P.C., P.Z. and T.T.T. conducted in vitro assays. B.P.C., S.J.P. and M.J.B. processed the cryo-EM data, built the model and performed refinement. S.J.P. and M.J.B. performed multivariate analysis, assisted in data interpretation and assisted in figure preparation. X.L. performed NMR measurements and analyzed spectra. R.D. prepared the cryo-EM samples and collected EM data. P.M.S., D.W. and S.H.G. supervised the project. B.P.C., P.M.S., D.W. and S.H.G. interpreted data, generated figures and wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Patrick M. Sexton, Denise Wootten or Samuel H. Gellman.

Ethics declarations

Competing interests

S.H.G. is a cofounder of Longevity Biotech, Inc., which is pursuing biomedical applications for α/β-peptides. P.M.S. receives research funding from Laboratoires Servier in the area of GPCR drug discovery. The current study is 100% independent from all academic or commercial collaborations with industry.

Additional information

Peer review information Nature Chemical Biology thanks Yan Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 G protein conformation assay time-courses.

The ligand induced BRET is baseline subtracted. Agonist was added at time 2 min and GTP (30 µM) was added after the 12.1 minute timepoint. The values indicated by the colored keys indicate concentrations (log[peptide (M)]). Data points represent the mean of at least three independent experiments. n = 3, 7, 7, 4, 4, and 3 independent replicates for GLP-1, Ex4, Ex4-D-Ala, Ex4-R,R-X, Ex4-L-Ala, and Ex4-S,S-X, respectively. Error bars represent standard error.

Source data

Extended Data Fig. 2 Purification and characterization of GLP-1R/Ex4-D-Ala/DNGas/Gβ1/Gγ2/Nb35 complex.

(a) Size-Exclusion chromatogram of crude anti-FLAG elution. (b) Fluorescence-detected size-exclusion chromatograph of purified complex. (c) Western blot. The channel corresponding to anti-His6 antibody is depicted in blue. The channel corresponding to anti-FLAG antibody channel is depicted in green. The channel corresponding to anti-Gas antibody is depicted in red. (d) Coomassie-stained, reducing SDS-PAGE gel. The lanes labeled 1, 2, and 3 correspond to LMNG/CHS solubilized fraction, anti-FLAG column flow-through, and purified complex, respectively for both (c) and (d). (e) Representative 2D-classes from negative-stain, single-particle electron microscopy of GLP-1R/Ex4-D-Ala/DNGas/Gβ1/Gγ2/Nb35 complex. Purification and characterization of GLP-1R/Ex4-S,S-X/DNGas/Gβ1/Gγ2/Nb35 complex. (f) Size-Exclusion chromatogram of crude anti-FLAG elution. (g) Fluorescence-detected size-exclusion chromatograph of purified complex. (h) Western blot. The channel corresponding to anti-His6 antibody is depicted in blue. The channel corresponding to anti-FLAG antibody channel is depicted in green. The channel corresponding to anti-Gas antibody is depicted in red. (i) Coomassie-stained, reducing SDS-PAGE gel. The lanes labeled 1, 2, 3, and 4 correspond to LMNG/CHS solubilized fraction, anti-FLAG column flow-through, anti-FLAG elution, and purified complex, respectively for both (c) and (d). (j) Representative 2D-classes from negative-stain, single-particle electron microscopy of GLP-1R/Ex4-S,S-X/DNGas/Gβ1/Gγ2/Nb35 complex. N = 2 biological replicates for c, d, (representative data) and N = 1 for h, and i.

Extended Data Fig. 3

An overview of the cryo-EM data processing pipeline for the Ex4-D-Ala/GLP-1R/Gs complex.

Extended Data Fig. 4 CryoEM map reconstructions.

(a, e, j) Gold-standard Fourier Shell Correlation (FSC) curves for the consensus map (a), conformer 1 (e), and conformer 2 (j) showing overall nominal resolutions of 2.3 Å, 2.4 Å, and 2.5 Å, respectively. Black, green, blue and red curves indicate corrected, unmasked, masked, and phase-randomized maps, respectively. (b, f, k) Euler angle distribution histograms of the particles used in reconstructions for the consensus map (B), conformer 1 (f), and conformer 2 (k). (c-d,g-i,l-n) Local resolution estimates shown as colored heatmaps. High threshold maps with resolution-estimate heatmaps for the consensus map (c), conformer 1 (g), and conformer 2 (l). Low threshold maps with resolution-estimate heatmaps for the consensus map (d), conformer 1 (h), and conformer 2 (m). Receptor-focused refinment maps for conformer 1 (i) and conformer 2 (n).

Extended Data Fig. 5 Map to model figures for selected features of Conformer 1 (a) and Conformer 2 (b).

Residues are show in paratheses. a indicates the map threshold.

Extended Data Fig. 6 Analysis of Ex4-D-Ala bound to GLP-1R.

(a) A Ligplot+ v2.2 diagram of the N-terminal interacting residues of Ex-4-D-Ala bound to GLP-1R as observed in the conformer 1 model. Residues of the agonist are indicated by the chain (P) denotation and GLP-1R residues are indicated by the (R) denotation. Hydrophobic interactions are shown with solid red lines, ionic interactions are show with dotted red lines, and hydrogen bonding is indicated with dotted green lines. Hydrogen bonds are shown with a maximum distance of 3.35 A and other non-bonded contacts are shown with 3.90 A. A comparison of GLP-1R structures with ligand removed (and ECD removed in top views) for clarity. (b) Side view and (c) top view of GLP-1R bound to TT-OAD2, PF-06882961, and Ex-4-D-Ala’s two conformers shown in purple, yellow, blue and orange, respectively. (d) Side view and (e) top view of GLP-1R bound to Ex-P5, Chu-128 and Ex-4-D-Ala’s two conformers shown in cyan, brown, blue, and orange, respectively.

Extended Data Fig. 7 Receptor-focused refinement of conformer 2.

The receptor-focused density map of Ex4-D-Alaconf. 2 with the model of exendin-9-39-bound to the GLP-1R ECD (PDB: 3C5T) docked to the density. b, A close view showing density for selected side chains of the docked model shown in a. c, Comparison of the docked model from a,b overlaid with the Ex4-D-Alaconf. 1 model. d, A model of Ex4-D-Ala fitted (using ISOLDE) to the receptor-focused density map of Ex4-D-Alaconf. 2 with a helical n-terminus. e, A model of Ex4-D-Ala fitted (using ISOLDE) to the receptor-focused density map of Ex4-D-Alaconf. 2 with an extended n-terminus (orange), and a snapshot of the molecular dynamics simulations of Ex4-D-Ala with the receptor docked to the receptor-focused density map of Ex4-D-Alaconf. 2 showing an extended N-terminus (red).

Extended Data Fig. 8 Conformation of GLP-1 and analogs in solution.

(a-b) Far-UV Circular Dichroism peptide of peptides reconstituted in ultrapure water at 25 °C. All measurements were performed at 25 µM except GLP-1-L-Ala which was measured at a 14 µM. Inset graphs show a close-up of the region from 190-220 nm. (c-d) Far-UV Circular Dichroism peptide of peptides reconstituted in 30% (%v/v) of 2,2,2-trifluoroethanol (TFE) in ultrapure water at 25 µM concentration and 25 °C. (g) CαH chemical shift patterns for the N-terminal region of peptides. The residue number corresponds to number of residues from the N-terminus. ΔδCαH = δCαH(obs) - δCαH(RC), with δCαH(RC) values obtained from Wishart, et al. No value is shown for residue 1 because the N-terminal histidine residue is free, and no value is shown for position 4 due to the presence of unnatural substitutions.

Source data

Extended Data Fig. 9 Secondary structure analysis of Exendin-4 and analogs simulated in water.

(a) Left: Per-residue time course analysis of secondary structure during three simulations. Right: Per-residue averaged secondary structure observed. (b) The local effects of position-4 substitution on the fraction of helical secondary structure during simulations. (c) The local effects of position-4 substitution on the fraction of bend secondary structure during simulations.

Source data

Extended Data Fig. 10 Molecular dynamics simulations including the GLP-1R.

a, b, c. An outline of the MD simulations performed on the GLP-1R bound to Ex4-L-Ala (a), Ex4 (b) and Ex4-D-Ala (c). d, e, f. MD snapshots taken at frame 4000 (200 ns) of Ex4-L-Ala (a), Ex4 (c) and Ex4-D-Ala (e) bound to the GLP-1R from the selected simulated rebinding replicates. The GLP-1R is colored grey. Ex4-L-Ala, Ex4, and Ex4-D-Ala are colored green, white, and red, respectively. The Gαs C-terminal, h5 helix (residues 370-394) is colored yellow. g, h, i. Per-residue, secondary structure fractions for Ex4-L-Ala (g), Ex4 (h) and Ex4-D-Ala (i) bound to the GLP-1R averaged across five MD simulation replicates.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, Tables 1–6, video descriptions, methods and instrumentation.

Reporting Summary

Supplementary Video 1

Partial unbinding and binding simulation of Ex4-l-Ala. The MD trajectory is the merge of one metadynamics replica with one SuMD replica and 300 ns of classic MD. Ex4-l-Ala is represented as orange ribbon (backbone) and sticks (side chains, position 4 as thick stick), and GLP-1R is represented as white ribbon. GLP-1R side chains within 3 Å of Ex4-l-Ala are shown as cyan sticks. Red dotted lines indicate hydrogen bonds. The starting conformation of the backbone of Ex4-l-Ala (conformer 1) is shown in transparent orange.

Supplementary Video 2

Partial unbinding and binding simulation of Ex4. The MD trajectory is the merge of one metadynamics replica with one SuMD replica and 300 ns of classic MD. Ex4 is represented as orange ribbon (backbone) and sticks (side chains), and GLP-1R is represented as white ribbon. GLP-1R side chains within 3 Å of Ex4 are shown as cyan sticks. The starting conformation of the backbone of Ex4 is shown in transparent orange.

Supplementary Video 3

Partial unbinding and binding of Ex4-d-Ala. Hydrophobic interactions between Phe 9 and Y1521.47 and L1411.36 anchor the peptide to GLP-1R in both a conformer 1-like structure (beginning of the video) and a conformer 2-like structure (end of the video) during the simulation. The MD trajectory is the merge of one metadynamics replica with one SuMD replica and 300 ns of classic MD. Ex4-d-Ala is represented as orange ribbon (backbone) and sticks (side chains, position 4 as thick stick), and GLP-1R is represented as white ribbon. GLP-1R side chains within 3 Å of Ex4-d-Ala are shown as cyan sticks. Red dotted lines indicate hydrogen bonds. A model of the backbone of Ex4-d-Ala from the cryo-EM maps of GLP-1R conformer 2 is shown in transparent purple for reference.

Supplementary Video 4

A video summarizing cryoSPARC 3D variability analysis for the first three principal components of the consensus refinement.

Supplementary Video 5

Morphs between models generated by roughly fitting the Ex4-d-Ala–GLP-1R–Gαs complex to extreme frames from cryoSPARC 3D variability analysis. Supplementary Videos 2, 3 and 4 correspond to principle components 1, 2 and 3, respectively. Supplementary Video 2 does not include morphs for the peptide or extracellular domain because one extreme frame from component 1 showed poorly resolved density in these regions.

Supplementary Video 6

Same as Supplementary Video 5.

Supplementary Video 7

Same as Supplementary Video 5.

Source data

Source Data Fig. 1

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Source Data Fig. 2

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Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 8

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Source Data Extended Data Fig. 9

Statistical source data.

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Cary, B.P., Deganutti, G., Zhao, P. et al. Structural and functional diversity among agonist-bound states of the GLP-1 receptor. Nat Chem Biol 18, 256–263 (2022). https://doi.org/10.1038/s41589-021-00945-w

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