Molecular Complex of Cholecystokinin-8 and N-Terminus of the Cholecystokinin A Receptor by NMR Spectroscopy

Abstract

The bimolecular complex of the C-terminal octapeptide of cholecystokinin, CCK-8, with the N-terminus of the CCK(A)-receptor, CCK(A)-R(1-47), has been structurally characterized by high-resolution NMR and computational refinement. The conformation of CCK(A)-R(1-47), within the lipid environment used for the spectroscopic studies, consists of a well-defined alpha-helix (residues 3-9) followed by a beta-sheet stabilized by a disulfide linkage between C18 and C29, leading to the first transmembrane alpha-helix (TM1). Titration of CCK(A)-R(1-47) with CCK-8 specifically affects the NMR signals of W39 of the receptor, in a saturable fashion. This association is specific for CCK-8; no association was observed upon titration of CCK(A)-R(1-47) with other peptide hormones. The ligand/receptor complex was characterized by intermolecular NOEs between Tyr(27) and Met(28) of CCK-8 and W39 of CCK(A)-R(1-47). These findings suggest that CCK-8 binds to CCK(A) with the C-terminus within the seven-helical bundle and the N-terminus of the ligand, projecting out between TM1 and TM7, forming specific interactions with the N-terminus of the CCK(A) receptor. This mode of ligand binding, consistent with published mutagenesis studies, requires variation of the interpretation of recent findings from photoaffinity cross-linking studies.

Structural basis of cholecystokinin receptor binding and regulation

“…The critical functional roles of the sites of glycosylation of the CCK 1 receptor for proper folding and trafficking to the cell surface have been reported (Hadac et al, 1996). In addition to the conserved disulfide bond linking the first and second extracellular loops that is shared by CCK 1 and CCK 2 receptors, the CCK 1 receptor also has been shown to possess another intradomain disulfide bond within its amino terminus (Pellegrini & Mierke, 1999;Ding et al, 2003). Palmitoylation has also been directly demonstrated for the CCK 1 receptor (Miller, unpublished observation).…”

Structure–activity relationships of bifunctional cyclic disulfide peptides based on overlapping pharmacophores at opioid and cholecystokinin receptors

“…One hundred structures were generated and were refined using simulated annealing AMBER force field in the Discover module of Insight II. The iterative calculations were performed until distance violations did not exceed 0.16 Å. CCK-8 structure at CCK-A receptor was obtained from the Protein Databank with code 1D6G [40].…”

“…The stereoview of the lowest energy NMR structure of compound 1. Superimposed structures of compound 1 (blue) and CCK-8 (red) [40]. The two structures have similar turns along the backbone between residues 1 -4 and similar orientations of Tyr and Trp residues.…”

How does cholecystokinin stimulate exocrine pancreatic secretion? From birds, rodents, to humans

“…Mierke and colleagues (43) built a model of the human CCK1R. The TM motifs each form an ␣-helix that embeds into the lipid bilayer (42,107). There is a disulfide bond between C18 and C29 in the NH 2 terminus [CCK1R(1-47)] (107).…”