Craig Giragossian

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

A comparison of the conformational characteristics of the related hormones [Nle(15)] gastrin-17 and [Tyr(9)-SO(3)] 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 CCK(2) 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-NH(2). The C-terminal conformation of [Nle(15)] gastrin-17 contained a short alpha-helix spanning the Ala(11)-Trp(14) sequence and an inverse gamma-turn centered on Nle(15) while that of [Tyr(9)-SO(3)] cholecystokinin-15 contained a short 3(10) helix spanning its Met(10) to Met(13) sequence and an inverse gamma-turn centered on Asp(14). Significantly, both the C-terminal helices were found to terminate in type I beta-turns spanning the homologous Gly-Trp-Met-Asp sequences. This finding supports the hypothesis that this structural motif is a necessary condition for CCK(2) 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 [Nle(15)] gastrin-17 and [Tyr(9)-SO(3)] 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 CCK(1) activity and non-essential for CCK(2) activity, pointing to it as a possible differentiator in CCK(1)/CCK(2) receptor selection. The different tyrosyl orientations were retained in molecular models for the [Nle(15)] gastrin-17/CCK(2) receptor and [Tyr(9)-SO(3)] cholecystokinin-15/CCK(1) receptor complexes, highlighting the role of this residue as a likely CCK(1)/CCK(2) receptor differentiator.

Determination of ligand-receptor interactions of cholecystokinin by nuclear magnetic resonance

To date high resolution structural studies of G protein coupled receptors, with the exception of rhodopsin, have not been feasible using conventional spectroscopic techniques. To overcome these difficulties, the structural features of partial or intact domains of GPCRs have been studied by nuclear magnetic resonance spectroscopy and X-ray crystallography. Here, we describe the structural characterization of receptor domains from the cholecystokinin 1 and 2 receptors and the elucidation of intermolecular interactions between the extracellular receptor domains and CCK-8 by solution state nmr.

Conformational and molecular modeling studies of sulfated cholecystokinin-15

Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK (S(19)HRISDRD[SO(4)]-YMGWMDF(33)-NH(2)) were determined by NMR spectroscopy in a zwitterionic membrane-mimetic solvent system, composed of DPC micelles. The C-terminal octapeptide consisted of a well-defined pseudohelix that was nearly identical to the structure previously reported for nonsulfated CCK-8 in the same solvent system. N-terminal amino acids of CCK-15 were highly disordered, with no clear conformational preference. Extensive NOE-restrained molecular dynamics simulations of the CCK-15/CCK(1)-R complex suggested that almost all the experimentally determined intermolecular contact points provided by NMR, site-directed mutagenesis, and photoaffinity labeling could be simultaneously satisfied, when the N-terminus of the ligand is placed in close spatial proximity to the N-terminus of the receptor.

Structural characterization of lipopeptide agonists for the bradykinin B2 receptor

The conformational features of Pam-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PKD) and Pam-Gly(-1)-Lys(0)-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH (PGKD), the Pam-Lys and Pam-Gly-Lys analogues of bradykinin, have been determined by high-resolution NMR in a zwitterionic lipoid environment. Radical-induced relaxation of the (1)H NMR signals was used to probe the topological orientation of the peptides with respect to the zwitterionic lipid interface. The radical-induced relaxation and molecular dynamics (MD) data indicated that the palmitic acid and N-terminal amino acid residues embed into the micelles, while the rest of the polypeptide chain is closely associated with the water-micelle interface. Throughout the entire nuclear Overhauser effect restrained MD simulation, a nonideal type I beta-turn was observed in the C-terminus of PKD between residues 6 and 9, and a gamma-turn was observed in the C-terminus of PGKD between residues 6 and 7. Therefore, the additional glycine has a dramatic effect on the structural preferences of the biologically important C-terminus, an effect brought about by the interaction with the lipid environment. These structural features are correlated to the biological activity at the bradykinin B2 receptor.

Characterizing the Interactions Between Cholecystokinin and the G-Protein Coupled Receptors CCK 1 and CCK 2 : An NMR-Based Study

Cholecystokinin (CCK) is a peptide hormone and neurotransmitter involved in a number of physiological processes of the gall bladder and pancreas, in the gastrointestinal tract, and anxiety, memory, and analgesia in the CNS. These activities are elicited by activation of two distinct G-protein coupled receptors, CCK1 and CCK2. Our laboratory is involved in the structural characterization of the interaction of CCK-8 with the CCK1 and CCK2 receptors.