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 beta-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 Trp(48)-Asp(49)-Asn(50) 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.
Highlights
Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized
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
Additional experimentally derived constraints beyond the integrity of the peptide backbone are required to determine the correct orientation between these two receptor domains. We have generated such constraints by utilizing quantitative fluorescence resonance energy transfer (FRET)[3] measurements between four residues distributed throughout secretin as it is docked with this receptor and distinct extracellular residues located within the amino terminus and within each of the loops of this receptor
Summary
Full structural characterization of G protein-coupled receptors has been limited to rhodopsin, with its uniquely stable structure and ability to be crystallized. 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. This refined model of the intact receptor was fully consistent with the spatial approximation of the Trp48-Asp49-Asn[50] 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. Secretin Receptor FRET of molecular models of secretin docked to its receptor that were constrained to accommodate the proposed orientations between the amino terminus and helical bundle domains of receptors in this family (4 – 6)
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