Fluorescence resonance energy transfer analysis of secretin docking to its receptor: Mapping distances between residues distributed throughout the ligand pharmacophore and distinct receptor residues

Kaleeckal G. Harikumar, Polo C.H. Lam, Maoqing Dong, Patrick M. Sexton, Ruben Abagyan, Laurence J. Miller

Research output: Contribution to journalArticle

25 Scopus citations

Abstract

Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized. For other members of this important superfamily, direct structural insights have been limited to NMR structures of soluble domains. Two members of the Class II family have recently had the structures of their isolated amino-terminal regions solved by NMR, yet it remains unclear how that domain is aligned with the heptahelical transmembrane bundle domain of those receptors. Indeed, three distinct orientations have been suggested for different members of this family. In the current work, we have utilized fluorescence resonance energy transfer to establish the distances between four residues distributed throughout fully biologically active, high affinity analogues of secretin and distinct residues in each of four extracellular regions of the intact secretin receptor. These 16 distance constraints were utilized along with nine photoaffinity labeling spatial approximation constraints to study the three proposed orientations of the peptide-binding amino terminus and helical bundle domains of this receptor. In the best model, the carboxyl terminus of secretin was found to bind in a groove above the β-hairpin region of the receptor amino terminus, with its amino-terminal end adjacent to the third extracellular loop and top of transmembrane segment VI. This refined model of the intact receptor was also fully consistent with the spatial approximation of the Trp48-Asp49-Asn50 endogenous agonist segment with the third extracellular loop region that it has been shown to photolabel. This provides strong evidence for the orientation of peptide-binding and signaling domains of a prototypic Class II G protein-coupled receptor.

Original languageEnglish (US)
Pages (from-to)32834-32843
Number of pages10
JournalJournal of Biological Chemistry
Volume282
Issue number45
DOIs
StatePublished - Nov 9 2007

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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