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.
Department of Chemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Craig
C
Giragossian
Norbert
N
Schaschke
Luis
L
Moroder
Dale F
DF
Mierke
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