Calcitonin Receptor N-Glycosylation Enhances Peptide Hormone Affinity by Controlling Receptor Dynamics

Abstract

The class B G protein-coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N-glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin-bound, GlcNAc-bearing CTR ECD at 1.78 and 2.85 Å resolutions and analyze the mechanism of the glycan effect. The N130 GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone-bound, N-glycan-free ECD, which suggested that the GlcNAc might affect CTR dynamics not observed in the static crystallographic snapshots. Hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations revealed that glycosylation stabilized a β-sheet adjacent to the N130 GlcNAc and the N-terminal α-helix near the peptide-binding site while increasing flexibility of the peptide-binding site turret loop. These changes due to N-glycosylation increased the ligand on-rate and decreased its off-rate. The glycan effect extended to RAMP-CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N-glycosylation can modulate GPCR function by altering receptor dynamics.

Keywords: G protein-coupled receptor (GPCR); N-linked glycosylation; dynamic allostery; ligand binding kinetics; peptide hormone.

Figure 1.. Crystal structures of sCT bound to proximal GlcNAc-bearing CTR ECD

(A) The 1.78Å CTR ECD structure. (B) The 2.85Å CTR ECD structure. See Figure S1 for MBP-fused CTR ECD in the asymmetric unit cell. MBP is omitted for clarity. See Figure S2 for the electron density of N130 GlcNAc. (C and D) The detailed view of sCT interaction with CTR ECD. Hydrogen bonds are represented as a gray dotted line. See also Figure S4.

Figure 2.. Comparisons of the GlcNAc-bearing structures to the N-glycan free CTR ECD structure

(A) 1.78Å and (B) 2.85Å sCT-CTR ECD complexes were aligned with Mol A of the sCT-bound glycan-free CTR ECD from PDB ID 5II0. Mol B and Mol C of sCT-bound glycan-free CTR ECD were also aligned with sCT-bound glycan-free CTR ECD Mol A. See also Figure S4.

Figure 3.. Equilibrium and kinetic analyses of sCT binding to CTR ECD glycoforms

(A) Equilibrium saturation binding FP assay assessing binding of the indicated CTR ECD glycoforms to a FITC-sCT(22–32) probe. (B) Equilibrium FP competition binding assay assessing displacement of the probe by unlabeled sCT(22–32). FP assay was performed with duplicate samples at each receptor concentration using FITC-sCT(22–32) as a probe. Data points in the plot were shown as mean with SEM as error bars. When the error bars were shorter than the height of the symbol, the error bars were omitted. (C-E) sCT binding kinetics was measured with biolayer interferometry assay. Mean values of K_D, k_on, and k_off were shown. Representative curves of both FP and kinetic assays were shown from at least three independent experiments.

Figure 4.. N-glycosylation of CTR N130 alters ECD conformational dynamics

Selected CTR ECD residues corresponding to the peptide fragments were color-coded on the 2.85Å CTR ECD structure, and the deuterium uptake plots were provided. See Figure S6 for HDX results in the presence of excess sCT. See also Figure S7. HDX results were shown as mean ± SEM from at least three independent experiments. The statistically significant differences were analyzed by a Student’s t-test (*p < 0.05).

Figure 5.. Molecular dynamics (MD) simulations with CTR ECD with or without GlcNAcs

(A) RMSF (Root Mean Squared Fluctuation) for the dynamics of the GlcNAc-bearing 1.78Å CTR ECD and glycan-free CTR ECD (PDB 5II0). Residues 44–136 were used for all MD simulations. Averaged RMSF values were shown from two molecules of the 1.78Å structure and three molecules of glycan-free CTR ECD present in the asymmetric unit. (B) RMSF for the dynamics of the GlcNAc-removed 1.78Å CTR ECD and glycan-free CTR ECD (PDB 5II0). (C-F) RMSF of CTR ECD was visualized using VMD showing five representative frames during the MD simulations. Two end frames of each direction were colored with blue or red, and the middle frame was colored with gray.