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Allosteric interactions in the parathyroid hormone GPCR–arrestin complex formation

Nature Chemical Biology volume 16, pages 1096–1104 (2020)Cite this article

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Abstract

Peptide ligands of class B G-protein-coupled receptors act via a two-step binding process, but the essential mechanisms that link their extracellular binding to intracellular receptor–arrestin interactions are not fully understood. Using NMR, crosslinking coupled to mass spectrometry, signaling experiments and computational approaches on the parathyroid hormone (PTH) type 1 receptor (PTHR), we show that initial binding of the PTH C-terminal part constrains the conformation of the flexible PTH N-terminal signaling epitope before a second binding event occurs. A ‘hot-spot’ PTH residue, His9, that inserts into the PTHR transmembrane domain at this second step allosterically engages receptor–arrestin coupling. A conformational change in PTHR intracellular loop 3 permits favorable interactions with β-arrestin’s finger loop. These results unveil structural determinants for PTHR–arrestin complex formation and reveal that the two-step binding mechanism proceeds via cooperative fluctuations between ligand and receptor, which extend to other class B G-protein-coupled receptors.

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Fig. 1: NMR analysis uncovers molecular aspects of the PTH–PTHR binding mechanism.
Fig. 2: Expanded two-step binding mechanism.
Fig. 3: PTHH9A dramatically alters PTHR signaling.
Fig. 4: PTHH9A stabilizes a unique receptor conformation.
Fig. 5: Identification of key contacts in the PTHR–β-arrestin complex interface by photo-crosslinking coupled to MS in live cells.

Data availability

All materials, data, and associated protocols will be made available to all qualified investigators from the corresponding authors upon reasonable request or with a simple institutional material transfer agreement. Source data are provided with this paper.

Code availability

MS raw data have been deposited to MASSIVE (ftp://massive.ucsd.edu/MSV000084971/) under the project name ‘Allosteric interactions in the parathyroid hormone class B GPCR–arrestin complex formation’. Codes for NMR and MD simulations in this study are available from corresponding authors upon reasonable request.

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Acknowledgements

Research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Disease, the National Institute of General Medical Sciences, the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the National Institute on Drug Abuse of the US National Institutes of Health under grant awards nos. R01-DK111427, R01-DK116780 and R01-DK122259 (to J.-P.V.); DK011794 (to T.J.G.); P41-GM103712 and P30-DA035778 (to I.B.); R01-DA046939 (P.T.); and F31-AR074843 (to L.J.C.).

Author information

Author notes

  1. These authors contributed equally: Lisa J. Clark, James Krieger.

Authors and Affiliations

  1. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Lisa J. Clark, Alex D. White, Saifei Lei, Fei Fang, Frederic Jean-Alphonse, Pei Tang, Kunhong Xiao, Ieva Sutkeviciute & Jean-Pierre Vilardaga

  2. Graduate Program in Molecular Biophysics and Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Lisa J. Clark

  3. Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    James Krieger, Ji Young Lee, Pemra Doruker, Pei Tang & Ivet Bahar

  4. Graduate Program in Molecular Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Alex D. White

  5. Department of Anesthesiology and Perioperative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

    Vasyl Bondarenko & Pei Tang

  6. Institute for Biochemistry, University of Leipzig, Leipzig, Germany

    Thore Böttke & Irene Coin

  7. Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    Thomas J. Gardella

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  1. Lisa J. Clark
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Contributions

L.J.C. performed and analyzed NMR studies with V.B. and P.T., and MD simulations with J.K., P.D. and I.B. J.Y.L. performed ANM calculations. A.D.W. performed signaling studies. T.B. and I.C. generated arrestin mutants and developed photo-crosslinking assays. I.S., F.J.-A., S.L., F.F. and K.X. optimized and performed photo-crosslinking coupled to MS assays. I.S. designed and optimized purification for the PTH(1–34) construct. T.J.G. provided guidance for radioligand binding studies. J.-P.V. was responsible for the overall conceptual composition and supervision of the study in collaboration with I.B., and wrote the manuscript with L.J.C. and I.S.

Corresponding authors

Correspondence to Ivet Bahar or Jean-Pierre Vilardaga.

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Extended data

Extended Data Fig. 1 Two-dimensional 1H-15N transverse relaxation optimized spectroscopy (TROSY) spectra of 15N-PTH(1–34).

Spectra were obtained in the absence (blue) or presence of PTHRECD, 0.5 (pink) or 0.75 (purple) molar ratio. a, On the left panel, full spectra showing all peaks of PTH(1–34). On the right panel, spectra of Gly12 and Ser17. Ser17 peak was too weak to be seen at 0.75 molar ratio. b, Zoomed-in spectra showing most peaks of PTH(1–34). PTH(1–34) peaks are labeled with bold text, and peaks corresponding to attached Strep-tag are labeled with italicized text. c, Zoomed-in spectral region of PTH His9. The conformation of His9 in free PTH is marked by Peak #1. Peak #2 represents the new conformation of His9 that occurred in the presence of 0.5 molar ratio PTHRECD and becomes stronger at 0.75 molar ratio PTHRECD. d, Zoomed-in spectral region of PTH Asn16, showing PTH conformational changes in the presence of PTHRECD. Peak #1 represents the conformation of Asn16 in the free PTH. Peak #2 represents the new conformation of Asn16 when PTH is bound to PTHRECD.

Source data

Extended Data Fig. 2 TROSY peak intensity (height), peak intensity ratio (Ibound/Ifree), and chemical shift perturbation (Δδ).

a, Peak intensities (normalized with respect to the number of scans in TROSY experiments) varied along the PTH sequence and the variations increased upon binding to 0.5 and 0.75 molar ratios of PTHRECD due to reduced PTH flexibility. b, Peak intensity ratio (Ibound/Ifree) and c, Chemical shift perturbation (Δδ) of individual residues upon binding to 0.5 or 0.75 molar ratios of PTHRECD. Residue intensities from the same experiment were normalized with respect to the intensity of Ser3 (indicated by an arrow) prior to calculations in b. Residues 15–17, indicated by asterisks, do not have visible peaks in the presence of 0.75 molar ratio PTHRECD. The new peak at Asn16 in the presence of 0.75 molar ratio PTHRECD was not used for Ibound/Ifree analyses but is displayed in the chemical shift perturbation plot. PTH(1–34) is separated from residues in the linker and Strep-tag by a thin dashed line.

Source data

Extended Data Fig. 3 Docking of PTH onto PTHRECD in the apo state using conformers from MD.

The NMR structural ensemble of free PTH(1–34) (cyan, PDB 1ZWA) was aligned onto MD snapshots of apo PTHR (dark green, see Methods for docking protocol). When the PTH ensemble is aligned onto PTHRECD oriented toward PTHRTMD, the N-terminal portion of PTH notably clashes with extracellular loops 1 and 2 (ECL1 and ECL2, colored purple and light orange, respectively). These clashes are reduced when the PTH ensemble is aligned onto PTHRECD oriented away from PTHRTMD.

Extended Data Fig. 4 Binding, trafficking and signaling properties of PTHH9A.

a, Co-immunoprecipitation of HA-tagged PTHR and β-arrestins from HEK293 cells after stimulation with PTHWT. Stimulation with PTHH9A does not immunoprecipitate β-arrestin with PTHRHA. b, Averaged time courses of PTHR internalization after 60 s stimulation with 100 nM PTHWT or PTHH9A in HEK293 cells stably expressing PTHRSEP. Data represent the mean ± s.d. of N = 2 experiments with n = 18 cells per experiment (independent biological replicates). c, Radioligand competition assays of PTHWT and PTHH9A binding to PTHR R0 (G protein-free) conformation. d, Radioligand competition assays of PTHWT and PTHH9A binding to PTHR RG conformation. For c, d, data are averaged from N = 4 and N = 5 independent experiments, respectively. Square points and error bars represent mean ± s.e.m. Individual data points are also shown.

Source data

Extended Data Fig. 5 Interactions between PTHWT His9 side chain and PTHR residues, and comparison with the interactions of the mutant PTHH9A.

Panels a-e display MD snapshots of PTHWT-bound receptor, and panel f, those for the mutant H9A-bound receptor. PTHWT is cyan, and PTHR is green in panels a–e, and PTHH9A is light pink, and PTHR is hot pink in panel f. MD snapshots of PTHWT-bound receptor showing different interactions. a, His9 hydrogen bond with Tyr429 (ECL3). b, Hydrogen bonds with Ser355 and Gly357 main chain (ECL2). c, Hydrogen bond with Ser355 side chain (ECL2). d, Aromatic-aromatic interactions between His9 and Tyr429 (ECL3). e, Hydrogen bond with Gln364 (TM5). f, Interactions between PTHR Leu354 and PTHH9A Ala9 in triplicate simulations. MD snapshots were collected every 10 ns during the last 50 ns of each simulation are aligned by PTHRTMD (residues 180 to 460).

Extended Data Fig. 6 Receptor conformations stabilized by PTHWT and PTHH9A.

Snapshots of PTHWT- and PTHH9A-bound receptor collected during the last 50 ns of triplicate simulations (10 ns intervals), aligned by PTHR transmembrane helices. a, Aligned receptor structures, with dashed boxes indicating areas shown in b, c. The arrow points to the helical turn (residues 394 to 397) in ICL3. ECD (residues 27 to 179), ECL1 (residues 247 to 275), TM3 residues 276 to 280, and peptide residues 14 to 34 are hidden for clarity. b, ECL2 conformation. c, Left, relative inward movement of PTHH9A–PTHR TM5. Right, relative inward movement of PTHH9A–PTHR TM6. d, ICL3 conformations in PTHWT- and PTHH9A-bound receptor snapshots after one 200 ns simulation. The helical turn in ICL3 stabilized by PTHWT is indicated by an arrow.

Extended Data Fig. 7 PTHH9A-stabilized kink 5 and its consequences.

a, Thr392-Val455 distance distributions over triplicate 200 ns simulations of apo, PTHWT-bound, and PTHH9A-bound receptor. Raw data are shown as thin lines. Second-order smoothed data (over 30 neighbors) are shown as thick lines. Top, plots starting at y = 0. Bottom, plots starting at y = 20. b, Alignment of PTHH9A-bound receptor (hot pink) TMD with the TMD of the PTHR–Gs cryo-EM structure. Gαs is colored purple. Clashing residues are shown as sticks. c, Interaction of PTHR Arg3965.75 with β-arrestin-1. In the PTHWT-bound model (green), positively charged Arg396 engages with negatively charged Glu155 and Glu156 from β-arrestin-1. In the PTHH9A-bound model (hot pink), Arg396 is oriented away from β-arrestin Glu155-156, toward Arg312. Since arginine side chains are flexible, Arg396 in the PTHH9A-bound model would reorient away from Arg312.

Source data

Extended Data Fig. 8 Accelerated MD (aMD) of apo PTHR.

Two snapshots from 100 ns aMD simulation of apo PTHR were aligned by their transmembrane helices. The snapshot exhibiting the largest TM6 movement is colored dark green. The snapshot with the smallest TM6 displacement is colored yellow. a, Full receptor structures. b, Zoomed-in structures, with TM5, ICL3, and TM6 of each receptor indicated by colored arrows.

Extended Data Fig. 9 LC-MS/MS data identifying photo-crosslinking between PTHR and β-arrestin-1 F75Bpa in PTH-stimulated cells.

a, PTHR Thr392. b, PTHR Val384.

Extended Data Table 1 Binding to the R0 and RG states of the PTHR. Reported are ligand concentrations that inhibited binding of radioligand by 50% (IC50). Data are the mean value ± s.e.m. of n = 4 (R0 state), and n = 5 (RG state), each in duplicate. P values are pairwise comparison (Student’s t-test, two-sided) to PTH.
Full size table

Supplementary information

Supplementary Information

Supplementary Figs. 1–6 and Table 1.

Reporting Summary

Supplementary Video 1

Motion of PTHR in the presence of PTH. Receptor is shown in yellow (most of the structure) and red (from Arg390 to Leu481), and PTH is shown in green. Thr392 and Val455 are shown as black balls, and Leu354 and Ala9 as blue balls. The video illustrates the collective movement of the complex along ANM soft modes 3, 14 and 18.

Supplementary Data

Unprocessed blots for Supplementary Fig. 1b

Source data

Source Data Fig. 1, Extended Data Figs. 1 and 2

NMR data (peak assignments, chemical shifts, peak intensities and relevant calculations)

Source Data Fig. 2b

R.m.s.d. from MD simulations

Source Data Fig. 3

Statistical source data: (1) cAMP assay, (2) Barr recruitment

Source Data Fig. 4, Extended Data Fig. 7a

Statistical source data: (1) T392-V455 distances, (2) PTHR activation

Source Data Fig. 5a–c

Unprocessed western blots

Source Data Fig. 5d

Statistical source data: western blot band intensity plot

Source Data Extended Data Fig. 4

Statistical source data: (1) internalization assay, (2) binding assays

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Clark, L.J., Krieger, J., White, A.D. et al. Allosteric interactions in the parathyroid hormone GPCR–arrestin complex formation. Nat Chem Biol 16, 1096–1104 (2020). https://doi.org/10.1038/s41589-020-0567-0

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