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Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2 - PubMed

  • ️Fri Jan 01 2021

. 2021 Aug 10;118(32):e2101279118.

doi: 10.1073/pnas.2101279118.

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Molecular insights into differentiated ligand recognition of the human parathyroid hormone receptor 2

Xi Wang et al. Proc Natl Acad Sci U S A. 2021.

Abstract

The parathyroid hormone receptor 2 (PTH2R) is a class B1 G protein-coupled receptor (GPCR) involved in the regulation of calcium transport, nociception mediation, and wound healing. Naturally occurring mutations in PTH2R were reported to cause hereditary diseases, including syndromic short stature. Here, we report the cryogenic electron microscopy structure of PTH2R bound to its endogenous ligand, tuberoinfundibular peptide (TIP39), and a heterotrimeric Gs protein at a global resolution of 2.8 Å. The structure reveals that TIP39 adopts a unique loop conformation at the N terminus and deeply inserts into the orthosteric ligand-binding pocket in the transmembrane domain. Molecular dynamics simulation and site-directed mutagenesis studies uncover the basis of ligand specificity relative to three PTH2R agonists, TIP39, PTH, and PTH-related peptide. We also compare the action of TIP39 with an antagonist lacking six residues from the peptide N terminus, TIP(7-39), which underscores the indispensable role of the N terminus of TIP39 in PTH2R activation. Additionally, we unveil that a disease-associated mutation G258D significantly diminished cAMP accumulation induced by TIP39. Together, these results not only provide structural insights into ligand specificity and receptor activation of class B1 GPCRs but also offer a foundation to systematically rationalize the available pharmacological data to develop therapies for various disorders associated with PTH2R.

Keywords: G protein–coupled receptor; cryo-electron microscopy; ligand recognition; parathyroid hormone receptor 2; syndromic short stature.

Copyright © 2021 the Author(s). Published by PNAS.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.

The overall cryo-EM structure of the TIP39–PTH2R–Gs complex. (A) Cut-through view of the cryo-EM density map that illustrates the TIP39–PTH2R–Gs complex and the disk-shaped micelle. The unsharpened cryo-EM density map at the 0.06 threshold shown as gray surface indicates a micelle diameter of 11 nm. The colored cryo-EM density map is shown at 0.12 threshold. (B) Model of the complex as a cartoon, with TIP39 as helix in green. The receptor is shown in blue, Gαs in yellow, Gβ subunit in cyan, Gγ subunit in navy blue, and Nb35 in gray. (C) The binding pocket of PTH2R accommodates peptide ligands of class B1 receptors. TIP39 is compared with LA-PTH (Left), GLP-1 (Middle), and glucagon (Right), respectively

Fig. 2.
Fig. 2.

Molecular recognition and ligand specificity of PTH2R. (A) Overall contacts between PTH2R (blue) and TIP39 (green). (B) Detailed contacts between PTH2R (blue) and TIP39 (green) within the ECD or the TMD. Key residues are shown as sticks. (C) Effects of receptor mutations on TIP39-induced cAMP accumulation. Data shown are means ± SEM of at least three independent experiments.

Fig. 3.
Fig. 3.

Ligand specificity between PTH1R and PTH2R. (A) Structural comparison of TIP39–PTH2R–Gs and LA-PTH–PTH1R–Gs complexes. Receptor ECD and G protein are omitted for clarity. (B) Schematic diagram of interactions between peptide and receptor. Conserved residues in PTH1R and PTH2R are highlighted in pink, while those similar are shown in light pink. Amino acid residues of peptides are colored red, negatively charged; blue, positively charged; yellow, hydrophilic; green, aromatic; gray, hydrophobic. Hydrophobic contacts are omitted for clarity. (C) Representative snapshots from MD simulations showing the key residues that determine the ligand specificity of PTH2R (blue). TIP39, PTH, and PTHrP are depicted in green, light green, and pink, respectively.

Fig. 4.
Fig. 4.

Molecular mechanism of the TIP(7-39) antagonism at PTH2R. (A) Sequence alignment between TIP39 and TIP(7-39). (B) A representative snapshot from the TIP39-bound PTH2R simulation system showing a TIP39-induced conformational change of the TM6 helix. (C) A representative snapshot from the TIP(7-39)-bound PTH2R simulation system showing a TIP(7-39)-induced conformational change of the TM6 helix. (D) A representative snapshot from the TIP39-bound PTH2R simulation system showing the N terminus of TIP39 insertion between TM5 and TM6 helices. Key residues are shown as sticks. Hydrogen bonds are shown as dashed lines. The Cα atoms of residues Ile3265.47b and Ile3776.54b are shown as spheres. (E) A representative snapshot from the TIP(7-39)-bound PTH2R simulation system showing a conformational change of the TM6 helix. (F) Frequency of a PTH2R residue interacting with TIP39 (green) or TIP(7-39) (yellow) in simulations. The frequency value suggests the stability of a particular residue–peptide interaction. A large interacting frequency suggests a stable interaction.

Fig. 5.
Fig. 5.

G258D mutation disrupts the G protein-binding interface of PTH2R in MD simulations. (A) A representative snapshot from the WT PTH2R simulations. (B) Key interactions stabilizing the helical bundle of TM3, TM5, and TM6 in the WT PTH2R simulations. Key residues are shown as sticks. Gly2583.51b is shown as a yellow sphere. (C) A representative snapshot from the G258D PTH2R simulations. (D) Asp2583.51b disrupts the hydrophobic interactions among helices TM3, TM5, and TM6 in the G258D PTH2R simulations. Key residues are shown as sticks. Asp2583.51b is highlighted in yellow. (E) A representative conformation of the G protein–binding interface of the WT PTH2R in simulations. The cryo-EM structure of TIP39–PTH2R–Gs complex was aligned to the simulation model to show the position of G protein with respect to the receptor. (F) A representative conformation of the G protein-binding interface of the G258D PTH2R in simulations.

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