title: Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor.
abstract: Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We used cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin (sec), Phe(6), Thr(7), and Leu(10), and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, but had a lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys(6)-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys(7)-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys(10)-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.
authors.affiliation: From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259.
authors.forename: Maoqing
authors.initials: M
authors.lastname: Dong
authors.affiliation: Molsoft LLC, La Jolla, California 92037.
authors.forename: Polo C-H
authors.initials: PC
authors.lastname: Lam
authors.affiliation: Molsoft LLC, La Jolla, California 92037.
authors.forename: Andrew
authors.initials: A
authors.lastname: Orry
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville 3052, Australia, and.
authors.forename: Patrick M
authors.initials: PM
authors.lastname: Sexton
authors.affiliation: Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville 3052, Australia, and.
authors.forename: Arthur
authors.initials: A
authors.lastname: Christopoulos
authors.affiliation: Molsoft LLC, La Jolla, California 92037, the Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92037.
authors.forename: Ruben
authors.initials: R
authors.lastname: Abagyan
authors.affiliation: From the Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, miller@mayo.edu.
authors.forename: Laurence J
authors.initials: LJ
authors.lastname: Miller
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012633:Secretin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; C029937:secretin receptor; D012633:Secretin; D000242:Cyclic AMP; D003545:Cysteine
keywords: G protein-coupled receptor (GPCR); cell surface receptor; cyclic AMP (cAMP); gel electrophoresis; ligand-binding protein; membrane protein; molecular modeling; mutagenesis; peptides