ABSTRACT
The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs/chemistry , Molecular Dynamics Simulation , Receptors, G-Protein-Coupled/chemistry , Receptors, Gastrointestinal Hormone/chemistry , Secretin/chemistry , Amino Acid Sequence , Animals , Binding Sites/genetics , Cell Line , Cricetinae , Cryoelectron Microscopy , Crystallography, X-Ray , Cysteine/chemistry , Cysteine/metabolism , GTP-Binding Protein alpha Subunits, Gs/ultrastructure , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Insecta , Models, Molecular , Protein Binding , Protein Domains/genetics , Protein Structure, Secondary , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/ultrastructure , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Gastrointestinal Hormone/ultrastructure , Secretin/metabolismABSTRACT
125I-glucagon binding sites have been detected and quantified on the intestinal smooth muscle cells of the dog by means of ultrastructural autoradiographic methods. These binding sites are located mainly in the plasmalema. The present findings established a morphological correlation with the physiological data concerning the spasmolytic function of the glucagon on the intestinal wall.