G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors to determine their cryo-EM structures and complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and 3D variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound, CGRP receptor complex, our work provides important insight into mechanisms of class B1 GPCR activation.

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity–modifying proteins (RAMPs), AMY 1 R, AMY 2 R, and AMY 3 R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY 1 R with salmon CT (sCT), AMY 2 R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors. Class B1 GPCRs constitute a subfamily of 15 receptors that characteristically contain large extracellular domains (ECDs) and respond to long polypeptide hormones. Class B1 GPCRs are critical regulators of homeostasis, and as such, many are important drug targets. While most transmembrane proteins, including GPCRs, are recalcitrant to crystallization, recent advances in electron cryo-microscopy (cryo-EM) have facilitated a rapid expansion of the structural understanding of membrane proteins. As a testament to this success, structures for all the class B1 receptors bound to G proteins have been determined by cryo-EM in the past five years. Further advances in cryo-EM have uncovered dynamics of these receptors, ligands, and signalling partners. Here, we examine the recent structural underpinnings of the class B1 GPCRs with an emphasis on structure-function relationships.

Targeting amylin (Amy) receptors (AMYRs) can reduce body weight with additional benefits to other anti-obesity treatments such as glucagon-like peptide-1 receptor (GLP-1R) agonists. AMYRs are heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), yielding AMY1R, AMY2R and AMY3R, respectively. A hallmark of AMYR activation by Amy is the formation of a secondary structural motif, termed a "bypass motif" (residues S19-P25) that partly contributes to selective activation of cAMP responses at AMYRs over CTR. This study explored the feasibility of tuning the selectivity of Amy analogues by modifying the residues (19-22) located within the bypass motif, resulting in a selective AMYR agonist, San385, as well as a series of non-selective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, as well as San45-bound AMY3R and CTR, decoding the structure-activity relationship (SAR) of these peptides. In particular, San45 is conjugated at position 19 with a lipid modification that anchors the peptide at the edge of receptor bundle and enables an alternate binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. This unique mechanism provides a single intervention strategy through targeted lipid modification to the structure-based design of long-acting, nonselective, Amy-based DACRAs with potential anti-obesity effects.

Targeting amylin (Amy) receptors (AMYRs) can reduce body weight with additional benefits to other anti-obesity treatments such as glucagon-like peptide-1 receptor (GLP-1R) agonists. AMYRs are heterodimers of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), yielding AMY1R, AMY2R and AMY3R, respectively. A hallmark of AMYR activation by Amy is the formation of a secondary structural motif, termed a “bypass motif” (residues S19-P25) that partly contributes to selective activation of cAMP responses at AMYRs over CTR. This study explored the feasibility of tuning the selectivity of Amy analogues by modifying the residues (19-22) located within the bypass motif, resulting in a selective AMYR agonist, San385, as well as a series of non-selective dual amylin and calcitonin receptor agonists (DACRAs), with San45 being an exemplar. We determined the structure and dynamics of San385-bound AMY3R, as well as San45-bound AMY3R and CTR, decoding the structure-activity relationship (SAR) of these peptides. In particular, San45 is conjugated at position 19 with a lipid modification that anchors the peptide at the edge of receptor bundle and enables an alternate binding mode when bound to the CTR, in addition to the bypass mode of binding to AMY3R. This unique mechanism provides a single intervention strategy through targeted lipid modification to the structure-based design of long-acting, non-selective, Amy-based DACRAs with potential anti-obesity effects.