Molecular Determinants for Ligand Selectivity of the Cell-Free Synthesized Human Endothelin B Receptor

• Published in: Journal of molecular biology 2018-12, Vol.430 (24), p.5105-5119
• Main Authors: Dong, Fang, Rues, Ralf B., Kazemi, Sina, Dötsch, Volker, Bernhard, Frank
• Format: Article
• Language: English
• Subjects: cell-free expression; endothelin receptors; GPCR; ligand selectivity; nanodiscs
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2018.10.006

Extracellular domains of G-protein-coupled receptors act as initial molecular selectivity filters for subtype specific ligands and drugs. Chimeras of the human endothelin-B receptor containing structural units from the extracellular domains of the endothelin-A receptor were analyzed after their co-translational insertion into preformed nanodiscs. A short β-strand and a linker region in the second extracellular loop as well as parts of the extracellular N-terminal domain were identified as molecular discrimination sites for the endothelin-B receptor-selective agonists IRL1620, sarafotoxin 6c, 4Ala-ET-1 and ET-3, but not for the non-selective agonist ET-1 recognized by both endothelin receptors. A proposed second disulfide bridge in the endothelin-B receptor tethering the N-terminal domain with the third extracellular loop was not essential for ET-1 recognition and binding, but increased the receptor thermostability. We further demonstrate an experimental approach with cell-free synthesized engineered agonists to analyze the differential discrimination of peptide ligand topologies by the two endothelin receptors. The study is based on the engineering and cell-free insertion of G-protein-coupled receptors into defined membranes and may become interesting also for other targets as an alternative platform to reveal molecular details of ligand selectivity and ligand binding mechanisms. [Display omitted] •Identification and modulation of molecular ligand selectivity filters in GPCRs.•Agonist binding to GPCRs can be controlled by directed engineering of small domains.•Different recognition mechanisms for the common agonist ET-1 by endothelin receptors•A second disulfide bond stabilizes endothelin B receptor.