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.
Highlights
The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) and its activation regulates calcium homeostasis and bone turnover [1, 2]
The human CTR extracellular domain (ECD) was fused with maltose-binding protein (MBP) at its N-terminus to facilitate crystallization [20] and a (His)6 tag at its C-terminus for purification
The fusion proteins were expressed as secreted proteins in HEK293 GnTI- cells [21] and the three glycosylation sites in the ECD, N73, N125, and N130 were trimmed to single GlcNAc residues with Endo H for crystallization
Summary
The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) and its activation regulates calcium homeostasis and bone turnover [1, 2]. CTR is a drug target for treating Paget’s disease, osteoporosis, and hypercalcemia, which all result from abnormal bone turnover and calcium regulation. A 32-amino acid peptide hormone salmon calcitonin (sCT), which has higher affinity than human calcitonin, has been developed as a drug for these clinical applications [3]. CTR can form a heterodimeric complex with any of three accessory Receptor Activity-Modifying Proteins (RAMPs), which enhance affinity for a 37-amino acid peptide hormone amylin [4,5,6]. Amylin is co-secreted with insulin from pancreatic β-cells and activates the CTR:RAMP complexes expressed in the brain [7, 8]. Understanding the molecular mechanism of how peptide hormone binding affinity is determined at CTR will provide an important foundational basis to develop peptide drugs targeting both CTR and the amylin receptors
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