Calcitonin Gene Related Peptide Receptor 1 (PDB ID=3N7R): from Homo sapiens
Created by Robert Wheeler
The calcitonin gene related peptide receptor 1 (CGRP-R) (PDB ID: 3N7R) is, as its name implies, a receptor for the calcitonin gene related peptide (CGRP). The two in combination act as a signaling pair implicated in migraines. CGRP is a found widely in the nervous system and is known as a potent vasodilator. Less is known about CGRP-R because it is a membrane bound protein that has few naturally abundant sources (1).
CGRP-R overall has a molecular weight of 48766.40 Da and exists physiologically as a heterodimer (2, 1). The heterodimer structure is common to class B receptors of the calcitonin like family of peptides. The other major receptors of the calcitonin family are generally formed by combinations of a receptor with a receptor activity-modifying protein (RAMP) subunit. Common primary receptors are the calcitonin receptor (CTR), and the calcitonin like receptor (CLR) which is sometimes called calcitonin receptor like receptor (3, 4). To date three RAMP proteins have been identified which have only 30% sequence identity to each other (5). In the case of CGRP-R a RAMP-1 combines with CLR and acts as a receptor for CGRP (1). Table 1 summarizes the combinations of CLR and CTR with the RAMP proteins, as well as the peptides each heterodimers show affinity towards (3).
The secondary structure of the CLR subunit of CGRP-R consists of a random coil leading into a α-helix comprised of residues 35-53. The structure returns to random coils after the helix before forming a finger-like structure between residues 65 and 82 which is stabilized by a disulfide bridge formed by Cys-48 and Cys-74. The finger motif eventually gives way to anti-parallel β-Sheets and the C terminus following yet another set of random coils (1). The secondary structure of the RAMP-1 of CGRP-R consists of a three helix bundle and a 3-10 helix where the α2 helix is antiparallel to α1 and α3 helix (6). The first alpha-helix runs from residues 28-51 and is broken by a kink caused by a disulfide bridge (1, 6). The next motifs the protein forms are a 3/10 helix between residues 53 and 56 before the second alpha-helix forms between residues 62 and 79. The final alpha-helix forms between residues 86 and 101(1). One interesting aspect of CGRP-R is that the RAMP-1 subunit does not change its shape upon binding the CLR subunit (1).
The heterodimer of CGRP-R forms through interactions primarily between the CLR α-helix and the α2 and α3 helix of RAMP-1 where the two sub-units are oriented perpendicularly to each other. The interaction is primarily via hydrogen bonds formed by the CLR’s side chains interaction with side chains on the RAMP amino acid backbone. Some specific interactions that create the heterodimer are hydrogen bonds, namely CLR’s Gln-54 attracting the hydrogen of RAMP’s Arg-102 and Cys-104. Another hydrogen bonding interaction is between CLR’s Gln-50 interacting with RAMP His-97, Phe-101, and Pro-105. Hydrophobic interactions also occur between the CLR Tyr-46 and RAMP Trp-59 and Phe-101, which helps stabilize the molecule (1).
Despite CGRP-R being uncommon in nature, the protein can be studied by synthetic means. A peptide of CGRP-R that was expressed by E. coli shows competency to bind with CGRP and common antagonists of CGRP. Recent studies have produced crystal structures of CGRP-R complexed with olcegepant and telecagepant antagonists of CGRP (1). The structures of both antagonists are shown in Figure 1.
CGRP-R binds differently with each antagonist, but the general tertiary and secondary structure of the CGRP-R is conserved. When CGRP-R binds with the antagonist, it forms a hydrogen bond with CLR Thr-122. The hydrogen bond interaction helps hold the antagonist in the hydrophobic binding pocket of the RAMP-1. The binding pocket consists of a ceiling composed of RAMP Trp-74 and Trp-84 and a “Trp Shelf” comprised of the side chain of Trp-72. When antagonists like olcegepant telecagepant bind Trp-72 rotates 70° relative to the unligand state closing the binding pocket. The piperdiene ring of olcegepant and telecagepant both π stack on the shelf, and a hydrogen bond forms with the amide of the indole ring (1).
The binding site is different for each of the antagonists, in order to accommodate the different inhibitory molecules. When the binding sites for the two antagonists discussed are superimposed it is found that the two have slightly different H-bonding sites and different hydrophobic interactions in the binding pocket. For example, whereas telecagepant displaces a water molecule resulting in a hydrophobic interaction with CLR Met-42, olecagepant relies on electrostatic interactions as well as hydrophobic interactions between its dibromotyrosyl group and the binding pocket (1).
Both a Dali and a BLAST search were run on CGRP-R to identify structurally similar proteins. A BLAST search compares sequences by assigning gaps in residues or groups of residues that are not different. Then by running a statistical test, an “E” value is assigned. E values range between 0 and infinity, and smaller E values indicate a greater similarity in sequence (7). The Dali search finds proteins of a similar tertiary structure of a given protein. Dali uses a sum of pairs method to determine the similarities in proteins and produces a Z for protein pairs. Proteins with a Z score greater than 2 are considered highly similar to reference protein (8).
The RAMP-2 domain was shown to have similar tertiary structure by a Dali search producing a Z score of 15.6, indicating a high degree of similarity (8). The similarity between RAMP-1 and RAMP-2 proteins is apparent when the two are superimposed. The main difference between the two proteins is that the α1 helix in RAMP-2 is straight whereas in the RAMP-1 the helix is kinked due to a disulfide bridge between Cys-27 and Cys-82. The two proteins both interact with the CLR subunit in the same way, using a hydrophobic patch on the α1 and α3 helices to interact with the α-helix of CLR (4).
Another combination of CLR and a RAMP protein were shown to have a high degree of structural similarity by a BLAST search. The protein labeled ADM1 (PDBID: 3AQF) in Table 1 consists of a combination CLR with RAMP-2 and produces an E value of 1e-53. ADM1 and CGRP-R are both very close to one another as both contain the same CLR domain. Due to different RAMP domains, the two molecules select for different peptides (7). CGRP-R specifically binds CGRP, and ADM1 specifically binds adrenomedullin, another molecule in the calcitonin family (3). Superpositon of the the CLR domains of ADM1 and CGRP-R reveals differences between the interface with the RAMP proteins. A prominent difference is the ADM1 binding pocket is less deep than the binding pocket of CGRP-R because the RAMP-2 protein is rotated and translated relative to RAMP-1 (4).
is a member of a large and varied family of CLR molecules which combine with different RAMP proteins to modify activity. CGRP-R forms a binding pocket between the CLR and RAMP-1 subunits which is defined by a Trp shelf and Trp ceiling and back. The pocket is used to bind two CGRP antagonist ligands olecagepant and telecagepant. The protein through the different interactions with ligands and subunits shows structure dictates the function of this protein.