Structural basis for extracellular interactions between calcitonin receptor‐like receptor and receptor activity‐modifying protein 2 for adrenomedullin‐specific binding

Abstract

The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

“…Our DsbC-assisted disulfide shuffling methodology is distinct from the traditional denaturant-based refolding protocols employed by other groups to produce CLR-RAMP ECD complexes. 17,27,37 We previously employed our methodology to produce several other class B GPCR ECDs with 3 disulfide bonds. [30][31][32][33][34] Here, the methodology was successful for proteins with up to 6 disulfide bonds, which suggests that it may be more broadly applicable to various disulfide bond-containing proteins.…”

“…27 Our results disagreed with the 70 nM K D reported for the binding of AM to a noncovalent RAMP2 ECD:CLR ECD complex as measured by SPR. 17 The basis for this discrepancy is unclear, but it seems unlikely that the ECD complex binds AM with nM affinity because several other class B GPCR ECD-peptide pairs exhibit lM binding affinities. [30][31][32][33]35 Although we were unable to obtain reliable ITC data for the tethered AM receptor ECD fusion, our AlphaScreen results nonetheless suggested that the MBP tag and covalent tether did not significantly alter AM binding.…”

“…Structural studies elucidated the folds of the CLR and RAMP1 and -2 ECDs and revealed how they interact in the absence of peptide ligands. [15][16][17] The CLR ECD fold is common to class B GPCR ECDs and consists of an N-terminal a-helix followed by a short consensus repeat fold that is composed of two b-sheets each with two b-strands. Three conserved disulfide bonds hold the secondary structure elements together.…”

“…28 Kusano et al demonstrated that recombinant CLR:RAMP2 ECD complex was a heterodimer at low concentration and a tetramer at high concentration, and their crystal structure of the complex showed a 1:1 heterodimer in the asymmetric unit and a 2:2 dimer of heterodimers formed by crystal symmetry. 17 Watkins et al reported the production and biochemical characterization of recombinant CLR:RAMP2 ECD complex with evidence for a 2:1 CLR:RAMP2 stoichiometry. 27 We previously described novel methodology for bacterial production of the AM receptor ECD complex as a maltose binding protein (MBP)-CLR ECD fusion protein in association with the RAMP2 ECD.…”

Preparation and Characterization of PEGylated Amylin

“…The protein was expressed in Escherichia coli BL21DE3pLys (1 mM IPTG, 37°C, 3 h) and purified as described elsewhere (27). In brief, RAMP2-ETD was produced in inclusion bodies.…”