Amylin is a peptide hormone co-expressed with insulin upon nutrient intake and its physiological role is to act as a satiation signal, slow gastric emptying and increase energy expenditure. Amylin's effects are mediated by amylin receptors (AMYRs), which are heterodimers composed of the class B GPCR calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMP1-3). There are many AMYR peptide agonists in clinical development for the treatment of metabolic diseases, most of which are based on the sequences of two endogenous peptides: rat amylin (rAmy) and salmon calcitonin (sCT). Although these two peptides are relatively well characterised pharmacologically, a detailed understanding of the molecular mechanisms by which they activate AMYRs is lacking. We sought to investigate the structural dynamics of AMY3R (CTR:RAMP3) in unbound (apo), rAmy bound and sCT bound states using hydrogen deuterium exchange mass spectrometry (HDX-MS).
We expressed AMY3R in insect cells, solubilised cell membranes in LMNG detergent and subsequently purified the receptors by affinity and size-exclusion chromatography for HDX-MS experiments. We achieved 83% and 84% sequence coverage for the CTR and RAMP3 respectively. Comparing the unbound and the two peptide bound states, we observed that both peptides reduced the deuterium uptake in both the CTR and RAMP3 extracellular domains, suggesting reduced overall dynamics in this region. Conversely, peptide binding appears to increase the dynamics of the RAMP3 transmembrane domain, evidenced by increased deuterium uptake in this region. Comparing the rAmy and sCT bound states, we observed several interesting differences. In transmembrane helix 6 (TM6) and extracellular loop 3 (ECL3) of CTR, sCT bound AMY3R had markedly reduced deuterium uptake compared to the rAmy bound state, particularly at later time points. Similarly, sCT conferred greater protection than rAmy at intracellular helix 8, which forms key contacts with G proteins. Taken together, our results provide molecular insights into the changes in dynamics that occur upon peptide binding to AMY3R, as well as a structural basis for the pharmacological profiles of rAmy and sCT.