title: 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).
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 acidp-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.
authors.affiliation: From the Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065.
authors.forename: Cassandra
authors.initials: C
authors.lastname: Koole
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Reynolds
authors.affiliation: the School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Mobarec
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia, and.
authors.forename: Caroline
authors.initials: C
authors.lastname: Hick
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia, and.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: From the Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, New York 10065, sakmar@mail.rockefeller.edu.
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Sakmar
mesh_terms: D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism; D003924:Diabetes Mellitus, Type 2 / Q000378:metabolism; D000077270:Exenatide; D000067757:Glucagon-Like Peptide-1 Receptor / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D000072417:Protein Domains; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays; D014688:Venoms / Q000737:chemistry*
chemical_list: D001386:Azides; C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D008024:Ligands; D010455:Peptides; D014688:Venoms; C027162:4-azidophenylalanine; D010649:Phenylalanine; D000077270:Exenatide; D000242:Cyclic AMP
keywords: G protein-coupled receptor (GPCR); genetic code expansion; glucagon-like peptide-1 receptor (GLP-1R); molecular modeling; mutagenesis; peptide hormone; photocross-linking; structural biology; structure-function; unnatural amino acid