☆ 4.7 Article

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

FASEB JOURNAL (2012)

期刊

FASEB JOURNAL

卷 26, 期 12, 页码 5092-5105

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FEDERATION AMER SOC EXP BIOL

DOI: 10.1096/fj.12-212399

关键词

class B GPCRs; molecular modeling; ligand binding

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Biochemistry & Molecular Biology Biology Cell Biology

资金

U.S. National Institutes of Health [DK46577]
National Health and Medical Research Council of Australia (NHMRC) [519461]
Peking Union Medical College Hospital (Beijing, China)

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摘要

While it is evident that the carboxylterminal 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 (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: 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. FASEB J. 26, 5092-5105 (2012). www.fasebj.org

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Allosteric communication regulates ligand-specific GPCR activity

Ning Ma, Anita K. Nivedha, Nagarajan Vaidehi

Summary: GPCRs are membrane-bound proteins that play a critical role in mediating multiple signaling pathways. They possess a dynamic nature and exist in an equilibrium between inactive and active states, with ligands selectively stabilizing specific conformations to shift the equilibrium. This ligand-selective effect is achieved through allosteric communication between the ligand binding site and G protein or beta-arrestin coupling site, allowing for the design of selective ligands and mutants with specific interactions.

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