Structure and dynamics of the CGRP receptor in apo and peptide-bound forms

Abstract

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.

Exploring Ligand Binding to Calcitonin Gene-Related Peptide Receptors

“…The current study focuses on the mechanism of binding for the N-terminal domain of CGRP that is critical to activation of CGRPR. Recent work elucidated the structures of apo and peptide-bound CGRPR in the absence of transducer protein ( Josephs et al, 2021 ) and revealed that, as expected, the peptide C-terminus is stably engaged with the ECD of the receptor, albeit that the ECD is highly dynamic. However, the N-terminus of the peptide only transiently engages with the core of the receptor.…”

“…The gradual engagement of the top of TM6, TM7 and ECL3 by the peptide N-terminal domain was accompanied by an inward movement of ECL3 ( Supplementary Video S1 ); ECL3 is important for CGRPR signaling ( Barwell et al, 2011 ) but its dynamics during receptor activation are still unclear. A comparison between the consensus maps of CGRP:CGRPR and CGRP:CGRPR:G s complexes ( Josephs et al, 2021 ) shows that the location of the top of TM6/7/ECL3 in the former structure partially overlaps with the location of the peptide binding pocket in the active CGRPR. Analysis of the conformational dynamics of the CGRP:CGRPR complex revealed that the top of TM6/7/ECL3 dynamically opens and closes in association with transient interaction of the peptide N-terminus and the TMD core ( Josephs et al, 2021 ).…”

“…A comparison between the consensus maps of CGRP:CGRPR and CGRP:CGRPR:G s complexes ( Josephs et al, 2021 ) shows that the location of the top of TM6/7/ECL3 in the former structure partially overlaps with the location of the peptide binding pocket in the active CGRPR. Analysis of the conformational dynamics of the CGRP:CGRPR complex revealed that the top of TM6/7/ECL3 dynamically opens and closes in association with transient interaction of the peptide N-terminus and the TMD core ( Josephs et al, 2021 ). This reinforces the concept that the presence of the G protein at the intracellular side of the receptor is needed to allosterically stabilize peptide N-terminal domain binding to the receptor core.…”

“…The heterodimer of CLR and RAMP1 displays selectivity for calcitonin gene-related peptide and therefore constitutes the prototypical CGRP receptor (CGRPR, Figures 1A,D ) ( Liang et al, 2018a ). The metastable binding of the neuropeptide CGRP to CGRPR facilitates the intracellular G protein engagement, which in turn allows the peptide to reach the final bound state ( Josephs et al, 2021 ). In this complex, CGRP inserts into the receptor TMD core via a disulfide-bridged N-terminal loop and short amphipathic α-helix, with an unstructured C-terminal tail extended towards the ECD, where it forms an additional anchoring point ( Figures 1A,D ).…”

“…In this complex, CGRP inserts into the receptor TMD core via a disulfide-bridged N-terminal loop and short amphipathic α-helix, with an unstructured C-terminal tail extended towards the ECD, where it forms an additional anchoring point ( Figures 1A,D ). The aforementioned stable binding mode can be achieved only after the engagement of the G protein ( De Lean et al, 1980 ; Chung et al, 2011 ; DeVree et al, 2016 ), which allosterically facilitates an outward movement of ECL3 and an upward shift of ECL2 ( Josephs et al, 2021 ). The CGRP-bound receptor, prior to G protein coupling, more closely resembles the apo structure with stable binding of the peptide C-terminus to the ECD, and where the peptide N-terminus only transiently and dynamically engaged with the receptor core ( Josephs et al, 2021 ).…”