Further evidence for a C-terminal structural motif in CCK2 receptor active peptide hormones

A comparison of the conformational characteristics of the related hormones [Nle15] gastrin-17 and [Tyr9-SO3] cholecystokinin-15, in membrane-mimetic solutions of dodecylphosphocholine micelles and water, was undertaken using NMR spectroscopy to investigate the possibility of a structural motif responsible for the two hormones common ability to stimulate the CCK2 receptor. Distance geometry calculations and NOE-restrained molecular dynamics simulations in biphasic solvent boxes of decane and water pointed to the two peptides adopting near identical helical C-terminal configurations, which extended one residue further than their shared pentapeptide sequence of Gly-Trp-Met-Asp-Phe-NH2. The C-terminal conformation of [Nle15] gastrin-17 contained a short α-helix spanning the Ala11-Trp14 sequence and an inverse γ-turn centered on Nle15 while that of [Tyr9-SO3] cholecystokinin-15 contained a short 310 helix spanning its Met10 to Met13 sequence and an inverse γ-turn centered on Asp14. Significantly, both the C-terminal helices were found to terminate in type I β-turns spanning the homologous Gly-Trp-Met-Asp sequences. This finding supports the hypothesis that this structural motif is a necessary condition for CCK2 receptor activation given that both gastrin and cholecystokinin have been established to follow a membrane-associated pathway to receptor recognition and activation. Comparison of the conformations for the non-homologous C-terminal tyrosyl residues of [Nle15] gastrin-17 and [Tyr9-SO3] cholecystokinin-15 found that they lie on opposite faces of the conserved C-terminal helices. The positioning of this tyrosyl residue is known to be essential for CCK1 activity and non-essential for CCK2 activity, pointing to it as a possible differentiator in CCK1/CCK2 receptor selection. The different tyrosyl orientations were retained in molecular models for the [Nle15] gastrin-17/CCK2 receptor and [Tyr9-SO3] cholecystokinin-15/CCK1 receptor complexes, highlighting the role of this residue as a likely CCK1/CCK2 receptor differentiator.