Molecular complex of cholecystokinin-8 and N-terminus of the cholecystokinin A receptor by NMR spectroscopy

The bimolecular complex of the C-terminal octapeptide of cholecystokinin, CCK-8, with the N-terminus of the CCKA-receptor, CCKA-R(1-47), has been structurally characterized by high-resolution NMR and computational refinement. The conformation of CCKA-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 (TMI). Titration of CCKA-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 CCKA-R(1-47) with other peptide hormones. The ligand/receptor complex was characterized by intermolecular NOEs between Tyr(27) and Met's of CCK-8 and W39 of CCKA-R(1-47). These findings suggest that CCK-8 binds to CCKA 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 CCKA receptor. This mode of ligand binding, consistent with published mutagenesis studies, requires variation of the interpretation of recent findings from photoaffinity cross-linking studies.