Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Bacterial expression and purification of a heterodimeric adrenomedullin receptor extracellular domain complex using DsbC-assisted disulfide shuffling.

Adrenomedullin (AM) is a peptide hormone that is a potent vasodilator and is essential for vascular development. The AM receptor is a heterodimeric cell surface receptor composed of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, in association with either of two receptor activity modifying protein (RAMP) coreceptors, RAMP2 or -3. The extracellular domains (ECDs) of CLR and the RAMPs form the primary AM binding site. Here, we present novel methodology for expression and purification of a heterodimeric AM receptor ECD complex as an MBP-CLR ECD fusion protein in association with the RAMP2 ECD. Co-expression of the RAMP2 ECD with the disulfide bond isomerase DsbC in the oxidizing cytoplasm of E. coli trxB gor enabled proper disulfide formation in vivo. The isolated RAMP2 ECD was purified to homogeneity. Co-expression of a soluble MBP-CLR ECD fusion protein with DsbC in E. coli trxB gor yielded a heterogeneous mixture of species with misfolded ECD. Incubation of affinity-purified MBP-CLR ECD in vitro with purified RAMP2 ECD, DsbC, and glutathione redox buffer promoted proper folding of the CLR ECD and formation of a stable MBP-CLR ECD:RAMP2 ECD complex that was purified by size-exclusion chromatography and which exhibited specific AM binding. Approximately 40mg of highly purified complex was obtained starting with 6L bacterial cultures for each protein. The methodology reported here will facilitate structure/function studies of the AM receptor.