Conformational and molecular modeling studies of beta-cyclodextrin-heptagastrin and the third extracellular loop of the cholecystokinin 2 receptor.
The conformational features of a conjugate of the C-terminus of human gastrin (HG[11-17]), the shortest gastrin sequence retaining biological function, with beta-cyclodextrin ([Nle(15)]-HG[11-17]-betaCD) were determined by NMR spectroscopy in an aqueous solution of dodecylphosphocholine (DPC) micelles. The peptide-betaCD conjugate displays a binding affinity and activation profile comparable to those of HG[11-17] at the cholecysokinin 2 (CCK(2)) receptor, the G protein-coupled receptor responsible for the gastrointestinal function of gastrin. The structure of the peptide consisted of a well-defined beta-turn between Gly(13) and Asp(16) of gastrin. The structural preferences of [Nle(15)]-HG[11-17]-betaCD in DPC micelles and the 5-doxylstearate-induced relaxation of the (1)H NMR resonances support a membrane-associated receptor recognition mechanism. Addition of [Nle(15)]-HG[11-17]-betaCD to the third extracellular loop domain of the CCK(2) receptor, CCK(2)-R(352-379), generated a number of intermolecular nuclear Overhauser enhancements (NOEs) and chemical shift perturbations. NOE-restrained MD simulations of the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex produced a topological orientation in which the C-terminus was located in a shallow hydrophobic pocket near the confluence of TM2 and -3. Despite the steric bulk and physicochemical properties of betaCD, the [Nle(15)]-HG[11-17]-betaCD-CCK(2)-R complex is similar to the CCK-8-CCK(2)-R complex determined previously, providing insight into the mode of ligand binding and the role of electrostatic interactions.
Giragossian
Craig
C
Department of Chemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Schaschke
Norbert
N
Moroder
Luis
L
Mierke
Dale F
DF
D000595:Amino Acid Sequence; D003198:Computer Simulation; D003505:Cyclodextrins / Q000737:chemistry*; D005110:Extracellular Space / Q000737:chemistry*; D005755:Gastrins / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043887:Receptor, Cholecystokinin B / Q000737:chemistry*; D055672:Static Electricity; D013816:Thermodynamics; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D005755:Gastrins; D008024:Ligands; D046911:Macromolecular Substances; D043887:Receptor, Cholecystokinin B; D047392:beta-Cyclodextrins; C037857:gastrin heptadecapeptide, Nle(15)-; C031215:betadex