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

• Published in: Journal of molecular biology 2020-03, Vol.432 (7), p.1996-2014
• Main Authors: Lee, Sang-Min, Jeong, Yejin, Simms, John, Warner, Margaret L., Poyner, David R., Chung, Ka Young, Pioszak, Augen A.
• Format: Article
• Language: English
• Subjects: BASIC BIOLOGICAL SCIENCES; Binding Sites; Crystallography, X-Ray; dynamic allostery; G protein-coupled receptor (GPCR); Glycosylation; Humans; ligand binding kinetics; Ligands; Models, Molecular; N-linked glycosylation; peptide hormone; Protein Binding; Protein Conformation; Protein Domains; Receptors, Calcitonin - chemistry; Receptors, Calcitonin - metabolism; Receptors, G-Protein-Coupled - metabolism; Signal Transduction
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2020.01.028

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. [Display omitted] •How N-glycosylation enhances the ligand affinity of the calcitonin receptor is unknown.•Crystal structures of calcitonin bound to N-glycosylated calcitonin receptor ECD.•MD simulations and HDX-MS experiments show that N-glycans modulate ECD dynamics.•N-glycans similarly enhance the ligand affinity of related amylin and CGRP receptors.•N-glycosylation of GPCRs can modulate their function by altering receptor dynamics.