Type II Turn of Receptor-bound Salmon Calcitonin Revealed by X-ray Crystallography

Abstract

Calcitonin is a peptide hormone consisting of 32 amino acid residues and the calcitonin receptor is a Class B G protein-coupled receptor (GPCR) , whereas CGRP and AM adopt type I turns. Our results suggest that a type II turn is the preferred conformation of calcitonin, whereas a type I turn is the preferred conformation of peptides that require RAMPs; CGRP, AM, and amylin. In addition the structure provides a detailed molecular explanation and hypothesis regarding ligand binding properties of CTR and the amylin receptors.. Calcitonin (CT)3 is a peptide hormone that possesses the ability to lower the calcium plasma concentration (1). CT is predominantly produced in C cells of the thyroid gland and it is secreted upon increases in the serum calcium concentration preventing hypercalcemia. Salmon CT (sCT) has been utilized for 40 years as a therapeutic agent for treatment of metabolic bone diseases such as osteoporosis and Paget's disease, due to its high potency; 40 -50 times higher compared with human CT. The calcitonin family of peptides also comprises amylin, adrenomedullin (AM), and two calcitonin gene-related peptides (␣-CGRP and ␤-CGRP) (2). These peptides all have an amino-terminal disulfide bond connecting residues 1 and 7 (residues 2 and 7 for amylin), a stretch of amphipathic ␣-helix, and an amide at the carboxyl-terminal residue.The CT receptor (CTR) is a Class B G protein-coupled receptor (GPCR) and it is expressed in many different cell types and tissues (3). Class B GPCRs are characterized by having a large ectodomain (ECD) of 100 -160 amino acid residues with a conserved tertiary structure stabilized by three conserved disulfide bridges. The ECD binds the carboxyl-terminal region of the peptide ligand and thereby positions the amino-terminal part of the ligand for binding to the transmembrane domain to initiate signaling, a mechanism referred to as the two-domain binding model (reviewed in Ref. 4). The structures of several Class B GPCR ECDs in complex with and without ligands have been determined including the ECDs of corticotropin-releasing factor receptor (5), pituitary adenylate cyclase-activating polypeptide receptor (6), glucose-dependent insulinotropic polypeptide receptor (7), parathyroid hormone-1 receptor (8), glucagon-like peptide-1 receptor (9), calcitonin receptor-like receptor (CLR) (10), and the glucagon receptor (11). The crystal structures of the transmembrane domains of the glucagon receptor (12) and the type 1 corticotropin releasing factor receptor (13) were determined recently but the structure of a full-length Class B receptor remains to be determined.The pharmacology of the CT family of peptides is complex due to heterodimerization of CTR or CLR with a membranebound receptor activity-modifying protein (RAMP). There are three different variants of RAMPs: RAMP-1, RAMP-2, and RAMP-3, which have ϳ30% amino acid sequence identity and the ligand specificity of CTR and CLR is determined by the interaction (or not) with the RAMPs. CTR is on its own the receptor for CT, whereas in comb...

N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity

“…5C). The similar affinities of these two peptides are consistent with previous findings that the 22-32 fragment constitutes the ECD binding region 14, 15 . Unfortunately, these low affinities make obtaining accurate K D values difficult because obtaining ideal sigmoid isotherms for the curve fitting would require high μM concentrations of ECD and peptide that are difficult and costly to obtain.…”

“…The crystal structure of sCT analog-bound N-glycan-free CTR ECD 15 was used to model the N-glycans at N73, N125, and N130 (Fig. 8A).…”

“…Studies over several decades by various groups yielded mixed results with no clear answer to the question of the role of N-glycosylation in the function of the class B GPCR CTR 14, 15, 17–19 . Defining the role of N-glycans can be challenging because it is often difficult to disentangle their effects on receptor biogenesis from effects on ligand binding and/or signaling, especially for intact receptors in cell-culture systems.…”

Phase-plate cryo-EM structure of a class B GPCR–G-protein complex

“…In the cryo-EM structure, the density corresponding to the ECD is lower in resolution than the rest of the complex (Extended Data Figure 4). Although this did not permit accurate modeling, there was a strong agreement with the isolated ECD/sCT structure (PDB: 5II0) 22 that could be fit to the density, contiguous with TM1 (Extended Data Figure 6). There is also additional density in the ECD around residue 130, corresponding to predicted glycosylation in this region.…”

“… a , Rigid body fitting of the structure of CTR ECD bound to sCT (PDB: 5II0) 22 into the corresponding regions of the cryo-EM map revealed additional density (close to residue 130) that may be attributed to glycosylation. b-d , Asp mutation of four consensus glycosylation residues (N28D, N73D, N125D and N130D) reveals little role of glycosylation on cell surface expression ( b ), determined via a cell surface ELISA to the N-terminal epitope tag.…”