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Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R)

Koole, Cassandra and Reynolds, Christopher A and Mobarec, Juan C and Hick, Caroline and Sexton, Patrick M and Sakmar, Thomas P (2017) 'Genetically encoded photocross-linkers determine the biological binding site of exendin-4 peptide in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R).' Journal of Biological Chemistry, 292 (17). 7131 - 7144. ISSN 0021-9258

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Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a key therapeutic target in the management of type II diabetes mellitus, with actions including regulation of insulin biosynthesis and secretion, promotion of satiety, and preservation of β-cell mass. Like most class B G protein-coupled receptors (GPCRs), there is limited knowledge linking biological activity of the GLP-1R with the molecular structure of an intact, full-length, and functional receptor·ligand complex. In this study, we have utilized genetic code expansion to site-specifically incorporate the photoactive amino acid p-azido-l-phenylalanine (azF) into N-terminal residues of a full-length functional human GLP-1R in mammalian cells. UV-mediated photolysis of azF was then carried out to induce targeted photocross-linking to determine the proximity of the azido group in the mutant receptor with the peptide exendin-4. Cross-linking data were compared directly with the crystal structure of the isolated N-terminal extracellular domain of the GLP-1R in complex with exendin(9–39), revealing both similarities as well as distinct differences in the mode of interaction. Generation of a molecular model to accommodate the photocross-linking constraints highlights the potential influence of environmental conditions on the conformation of the receptor·peptide complex, including folding dynamics of the peptide and formation of dimeric and higher order oligomeric receptor multimers. These data demonstrate that crystal structures of isolated receptor regions may not give a complete reflection of peptide/receptor interactions and should be combined with additional experimental constraints to reveal peptide/receptor interactions occurring in the dynamic, native, and full-length receptor state.

Item Type: Article
Uncontrolled Keywords: G protein-coupled receptor (GPCR); molecular modeling; mutagenesis; peptide hormone; structural biology; structure-function; glucagon-like peptide-1 receptor (GLP-1R); genetic code expansion; photocross-linking; unnatural amino acid
Subjects: Q Science > Q Science (General)
R Medicine > RM Therapeutics. Pharmacology
Divisions: Faculty of Science and Health > Life Sciences, School of
Depositing User: Christopher Reynolds
Date Deposited: 12 May 2017 13:22
Last Modified: 19 Aug 2019 21:15
URI: http://repository.essex.ac.uk/id/eprint/19576

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