Abstract
The activation mechanism of class B G-protein-coupled receptors (GPCRs) remains largely unknown. To characterize conformational changes induced by peptide hormones, we investigated interactions of the class B corticotropin-releasing factor receptor type 1 (CRF1R) with two peptide agonists and three peptide antagonists obtained by N-truncation of the agonists. Surface mapping with genetically encoded photo-crosslinkers and pair-wise crosslinking revealed distinct footprints of agonists and antagonists on the transmembrane domain (TMD) of CRF1R and identified numerous ligand-receptor contact sites, directly from the intact receptor in live human cells. The data enabled generating atomistic models of CRF- and CRF(12-41)-bound CRF1R, further explored by molecular dynamics simulations. We show that bound agonist and antagonist adopt different folds and stabilize distinct TMD conformations, which involves bending of helices VI and VII around flexible glycine hinges. Conservation of these glycine hinges among all class B GPCRs suggests their general role in activation of these receptors.
Highlights
We found that CRF1R peptide antagonists stabilize different conformations of the CRF1R transmembrane domain (TMD) in respect to the agonists, including large-scale movements of transmembrane helices and changes in the shape of the binding pocket
It has been largely believed that N-truncated peptide ligands of class B G-protein-coupled receptors (GPCRs) behave as antagonists because they occupy the binding site in the ECD, but are too short to reach the TMD, where activation takes place (Cordomi et al, 2017)
Our crosslinking results provide direct evidence that N-truncated antagonists of CRF1R extensively penetrate the TMD, which explains previous observations obtained with chimeric constructs and isolated receptor domains (Hoare et al, 2004, 2005), as well as the fact that the 30-mer antagonist Astressin induces CRF1R internalization (Perry et al, 2005)
Summary
G-protein-coupled receptors (GPCRs) of class B comprise a family of 15 transmembrane receptors that respond to endocrine factors and regulate vital functions in mammals, including glucose and calcium homeostasis, pain transmission and gastrointestinal regulation (Bortolato et al, 2014; Wootten et al, 2017; Culhane et al, 2015; Pal et al, 2012). The native ligands of class B GPCRs are long peptide hormones, which bear distinct functional sites at the two termini (Pal et al, 2012; Beyermann et al, 2000). Crystal structures have revealed the binding mode of ligand C-termini to the isolated ECDs of several class B
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