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

Dynamic activation of a GPCR G protein–coupled receptors (GPCRs) coordinate a complex information flow between the outside and inside of a cell. An increasing number of GPCR structures provide insight into function. However, the dynamics that link extracellular sensing to intracellular signaling are not completely understood, because GPCRs used in structure determination are generally modified to constrain their dynamics. Josephs et al. succeeded in determining the structures of an unmodified calcitonin gene–related peptide receptor, which is implicated in migraines, both alone and bound to its neuropeptide ligand. Based on the structures and data from complementary biophysical techniques, they show that initial binding of the peptide causes only minor conformational changes of the GPCR, but dynamically causes changes at the intracellular side that facilitate G protein binding and activation. Science, this issue p. eabf7258 Biophysics and cryo-EM elucidate how agonist binding facilitates calcitonin gene–related peptide receptor activation. INTRODUCTION G protein–coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Among these, class B1 GPCRs are activated by major peptide hormones and neuropeptides that play important roles in the physiological regulation of metabolic, cardiovascular, and immunological responses. Understanding both how natural and synthetic ligands bind to GPCRs and how agonists activate receptors to recruit transducer proteins is critical to understanding how the body regulates the flow of complex information that is presented to different cells and tissues to enable coordinated biological responses. RATIONALE Despite advances in structural biology, our understanding of the dynamic molecular processes that enable ligand binding and subsequent receptor activation and G protein recruitment is incomplete because of the difficulty in elucidating structural information for unmodified receptors in key intermediate states (e.g., apo and agonist-bound). To address the gap, we have applied single-particle cryo–electron microscopy (cryo-EM) to determine structures of these intermediate states for the calcitonin gene–related peptide (CGRP) receptor, a class B1 GPCR. The CGRP receptor, a key mediator in the pathology of migraines, is the primary target for the actions of the neuropeptide CGRP that has diverse physiological functions including the regulation of vascular tone and the modulation of inflammatory and metabolic responses. RESULTS After expression and purification of unmodified apo and peptide-bound CGRP receptors, we determined structures of these complexes using cryo-EM at global resolutions [Fourier shell correlation (FSC) = 0.143] of 3.2 and 3.5 Å, respectively. In the consensus structures, the intracellular face of both the apo and peptide-bound receptors are very similar, and both structures lacked the key hallmark features of activated class B1 GPCRs, including the large outward movement of the base of transmembrane helix 5 (TM5), TM6, and the interconnecting intracellular loop 3 (ICL3) that is required to accommodate G protein binding. Instead, the apo and CGRP-bound receptors exhibited a relatively straight TM6, and the base of TM5 and TM6 was packed closely with TMs 2, 4, and 7 and ICL2. Moreover, the CGRP N terminus that is required for receptor activation was not resolved, and only small differences in the static structures were observed between the apo and peptide-bound CGRP receptors. Consequently, we interrogated the conformational dynamics of both states of the receptor. Three-dimensional (3D) variance analysis of the cryo-EM data revealed that the CGRP N terminus formed only transient interactions with the receptor core that was more dynamic when the peptide was bound, and CGRP N-terminal engagement was correlated with the destabilization of ground-state interactions at the intracellular face of the receptor. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provided complementary data that supported the observations from the cryo-EM variance analysis. In combination with our previously published structure of the fully active CGRP-bound receptor in complex with the Gs protein, these results support a model of receptor activation whereby the apo receptor is in an inactive state that is stabilized by ground-state interactions at the base of the receptor. CGRP binds initially to the extracellular domain of the receptor, thereby increasing the dynamics of the extracellular face of the receptor that enables transient interaction of the CGRP N-terminal activation domain with the receptor core. Interactions of the peptide N terminus with the receptor core allosterically destabilize ground-state networks at the intracellular face of the receptor, which facilitates G protein binding with subsequent cooperative interactions between both the G protein and CGRP that drive the larger conformational changes required for full engagement of the G protein and stabilization of the binding of the peptide N terminus observed in the fully active structure. CONCLUSION Collectively, our work provides understanding of the structure and dynamics of the apo state of class B1 GPCRs and the mechanisms of receptor activation. The use of essentially unmodified receptor allowed us to interrogate the changes to conformational dynamics that occur upon agonist binding by complementary implementation of cryo-EM and HDX-MS, which provided insight into peptide agonist binding and the activation of the CGRP receptor. CGRP binding alters the dynamics of the CGRP receptor. HDX-MS and 3D variance analysis of the cryo-EM data provide strongly correlated data on the conformational dynamics of apo and CGRP-bound CGRPRs. The cryo-EM density map (starting frame) is shown in transparent gray surface representation. HDX-MS data are illustrated in licorice ribbon format, where the thickness and color correspond to the extent of deuterium uptake. RAMP1, receptor activity–modifying protein 1; ECD, extracellular domain; TM, transmembrane. G protein–coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a 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 from insect cells to determine their cryo–electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.