Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping

Maoqing Dong, Xiequn Xu, Alicja M. Ball, Joshua A. Makhoul, Polo C.H. Lam, Delia I. Pinon, Andrew Orry, Patrick M. Sexton, Ruben Abagyan, Laurence J. Miller

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

While it is evident that the carboxyl-terminal region of natural peptide ligands bind to the amino-terminal domain of class B GPCRs, how their biologically critical amino-terminal regions dock to the receptor is unclear. We utilize cysteine trapping to systematically explore spatial approximations among residues in the first five positions of secretin and in every position within the receptor extracellular loops (ECLs). Only Cys2 and Cys5 secretin analogues exhibited full activity and retained moderate binding affinity (IC50: 92±4 and 83±1 nM, respectively). When these peptides probed 61 human secretin receptor cysteine-replacement mutants, a broad network of receptor residues could form disulfide bonds consistent with a dynamic ligand-receptor interface. Two distinct patterns of disulfide bond formation were observed: Cys2 predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser340, 94±7%; Pro341, 84±9%; Phe258, 73±5%; Trp274 62±8%), and Cys5 labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln348, 100%; Ile347, 73±12%; Glu342, 59±10%; Phe351, 58±11%). These constraints were utilized in molecular modeling, providing improved understanding of the structure of the transmembrane bundle and interconnecting loops, the orientation between receptor domains, and the molecular basis of ligand docking. Key spatial approximations between peptide and receptor predicted by this model (H1-W274, D3-N268, G 4-F258) were supported by mutagenesis and residue-residue complementation studies.

Original languageEnglish (US)
Pages (from-to)5092-5105
Number of pages14
JournalFASEB Journal
Volume26
Issue number12
DOIs
StatePublished - Dec 1 2012

Keywords

  • Class B GPCRs
  • Ligand binding
  • Molecular modeling

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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    Dong, M., Xu, X., Ball, A. M., Makhoul, J. A., Lam, P. C. H., Pinon, D. I., Orry, A., Sexton, P. M., Abagyan, R., & Miller, L. J. (2012). Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping. FASEB Journal, 26(12), 5092-5105. https://doi.org/10.1096/fj.12-212399