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

Maoqing Dong, Polo C H Lam, Andrew Orry, Patrick M. Sexton, Arthur Christopoulos, Ruben Abagyan, Laurence J Miller

Research output: Contribution to journalArticle

6 Scopus citations

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), Phe6, Thr7, and Leu10, and cysteines incorporated into the extracellular face of the receptor. Each peptide wasafull agonist for cAMP, but had a lower binding affinity than natural hormone. These boundto 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 Cys6-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys7-sec predominantly labeling only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys10-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.

Original languageEnglish (US)
Pages (from-to)5172-5184
Number of pages13
JournalJournal of Biological Chemistry
Volume291
Issue number10
DOIs
StatePublished - Mar 4 2016

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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