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.
| Original language | English |
|---|---|
| Article number | e27711 |
| Journal | eLife |
| Volume | 6 |
| DOIs | |
| Publication status | Published - 3 Aug 2017 |
| Externally published | Yes |
Funding
This research was supported by the Emmy-Noether Program of the Deutsche Forschungsgemein-schaft (DFG) under grant CO822/2-1 to IC, the Netherlands Organization for Scientific Research (NWO) under Rubicon grant 019.161LW.035 to BZ, and by National Institute of Health grant U54 GM094618 to VK. We thank Lei Wang (UCSF) for sharing the library of CRF1R-TAG mutants previously prepared by IC and Tingting Sun at The Salk Institute for Biological Studies, Paul Sawchenko (Peptide Biology Laboratory, The Salk Institute) for sharing the anti-CRF and anti-Ucn1 antibodies prepared by Joan Vaughan, Bruce Branchini (Connecticut College) for sharing the PpyRE9 luciferase gene. We thank Torsten Schöneberg (University of Leipzig) for his help with the ELISA, and Angela Walker for help with preparing the manuscript. The molecular dynamics simulations described in this paper were conducted on the University of Southern California’s Center for High-Performance Computing clusters (hpcc.usc.edu).
| Funders | Funder number |
|---|---|
| Deutsche Forschungsgemein-schaft | CO822/2-1 |
| National Institute of Health | U54 GM094618 |
| Netherlands Organization for Scientific Research | |
| National Institute of General Medical Sciences | U54GM094618 |
| Nederlandse Organisatie voor Wetenschappelijk Onderzoek | 019.161LW.035 |