Spatial approximation between secretin residue five and the third extracellular loop of its receptor provides new insight into the molecular basis of natural agonist binding.
ABSTRACT The amino terminus of class II G protein-coupled receptors plays an important role in ligand binding and receptor activation. Understanding of the conformation of the amino-terminal domain of these receptors has been substantially advanced with the solution of nuclear magnetic resonance and crystal structures of this region of receptors for corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and gastric inhibitory polypeptide. However, the orientation of the amino terminus relative to the receptor core and how the receptor gets activated upon ligand binding remain unclear. In this work, we have used photoaffinity labeling to identify a critical spatial approximation between residue five of secretin and a residue within the proposed third extracellular loop of the secretin receptor. This was achieved by purification, deglycosylation, cyanogen bromide cleavage, and sequencing of labeled wild-type and mutant secretin receptors. This constraint has been used to refine our evolving molecular model of secretin docked at the intact receptor, which for the first time includes refined helical bundle and loop regions and reflects a peptide-binding groove within the receptor amino terminus that directs the amino terminus of the peptide toward the receptor body. This model is fully consistent with the endogenous agonist mechanism for class II G protein-coupled receptor activation, where ligand binding promotes the interaction of a portion of the receptor amino terminus with the receptor body to activate it.
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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 Cys(2) and Cys(5) secretin analogues exhibited full activity and retained moderate binding affinity (IC(50): 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: Cys(2) predominantly labeled residues in the amino terminus of ECL2 and ECL3 (relative labeling intensity: Ser(340), 94±7%; Pro(341), 84±9%; Phe(258), 73±5%; Trp(274) 62±8%), and Cys(5) labeled those in the carboxyl terminus of ECL2 and ECL3 (Gln(348), 100%; Ile(347), 73±12%; Glu(342), 59±10%; Phe(351), 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 (H(1)-W(274), D(3)-N(268), G(4)-F(258)) were supported by mutagenesis and residue-residue complementation studies.-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. Mapping spatial approximations between the amino terminus of secretin and each of the extracellular loops of its receptor using cysteine trapping.The FASEB Journal 09/2012; · 5.70 Impact Factor
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ABSTRACT: Understanding of the structural importance of each position along a peptide ligand can provide important insights into the molecular basis for its receptor binding and biological activity. This has typically been evaluated using serial replacement of each natural residue with an alanine. In the current report, we have further complemented alanine scanning data with the serial replacement of each residue within secretin-27, the natural ligand for the prototypic class B G protein-coupled secretin receptor, using a photolabile phenolic residue. This not only provided the opportunity to probe spatial approximations between positions along a docked ligand with its receptor, but also provided structure-activity insights when compared with tolerance for alanine replacement of the same residues. The pattern of sensitivity to phenolic residue replacement was periodic within the carboxyl-terminal region of this peptide ligand, corresponding with alanine replacements in that region. This was supportive of the alpha-helical conformation of the peptide in that region and its docking within a groove in the receptor amino-terminal domain. In contrast, the pattern of sensitivity to phenolic residue replacement was almost continuous in the amino-terminal region of this peptide ligand, again similar to alanine replacements, however, there were key positions in which either the phenolic residue or alanine was differentially preferred. This provided insights into the receptor environment of the portion of this ligand most critical for its biological activity. As the structure of the intact receptor is elucidated, these data will provide a guide for ligand docking to the core domain and to help clarify the molecular basis of receptor activation.Regulatory Peptides 11/2012; · 2.06 Impact Factor
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ABSTRACT: Class B guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) share heptahelical topology and signaling via coupling with heterotrimeric G proteins typical of the entire superfamily of GPCRs. However, they also exhibit substantial structural differences from the more extensively studied class A GPCRs. Even their helical bundle region, most conserved across the superfamily, is predicted to differ from that of class A GPCRs. Much is now known about the conserved structure of the amino-terminal domain of class B GPCRs, coming from isolated NMR and crystal structures, but the orientation of that domain relative to the helical bundle is unknown, and even less is understood about the conformations of the juxtamembranous amino-terminal tail or of the extracellular loops linking the transmembrane segments. We now review what is known about the structure and function of these regions of class B GPCRs. This comes from indirect analysis of structure-function relationships elucidated by mutagenesis and/or ligand modification and from the more direct analysis of spatial approximation coming from photoaffinity labeling and cysteine trapping studies. Also reviewed are the limited studies of structure of some of these regions. No dominant theme was recognized for the structures or functional roles of distinct regions of these juxtamembranous portions of the class B GPCRs. Therefore, it is likely that a variety of molecular strategies can be engaged for docking of agonist ligands and for initiation of conformational changes in these receptors that would be expected to converge to a common molecular mechanism for activation of intracellular signaling cascades.British Journal of Pharmacology 07/2013; · 5.07 Impact Factor