RT Journal Article SR Electronic T1 Refinement of the Conformation of a Critical Region of Charge-Charge Interaction between Cholecystokinin and Its Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1041 OP 1052 DO 10.1124/mol.61.5.1041 VO 61 IS 5 A1 Xi-Qin Ding A1 Delia I. Pinon A1 Kristina E. Furse A1 Terry P. Lybrand A1 Laurence J. Miller YR 2002 UL http://molpharm.aspetjournals.org/content/61/5/1041.abstract AB Insight into the molecular basis of cholecystokinin (CCK) binding to its receptor has come from receptor mutagenesis and photoaffinity labeling studies, with both contributing to the current hypothesis that the acidic Tyr-sulfate-27 residue within the peptide is situated adjacent to basic Arg197 in the second loop of the receptor. Here, we refine our understanding of this region of interaction by examining a structure-activity series of these positions within both ligand and receptor and by performing three-dimensional molecular modeling of key pairs of modified ligand and receptor constructs. The important roles of Arg197 and Tyr-sulfate-27 were supported by the marked negative impact on binding and biological response with their natural partner molecule when the receptor residue was replaced by acidic Asp or Glu and when the peptide residue was replaced by basic Arg, Lys, p-amino-Phe,p-guanidino-Phe, or p-methylamino-Phe. Complementary ligand-receptor charge-exchange experiments were unable to regain the lost function. This was supported by the molecular modeling, which demonstrated that the charge-reversed double mutants could not form a good interaction without extensive rearrangement of receptor conformation. The models further predicted that R197D and R197E mutations would lead to conformational changes in the extracellular domain, and this was experimentally supported by data showing that these mutations decreased peptide agonist and antagonist binding and increased nonpeptidyl antagonist binding. These receptor constructs also had increased susceptibility to trypsin degradation relative to the wild-type receptor. In contrast, the relatively conservative R197K mutation had modest negative impact on peptide agonist binding, again consistent with the modeling demonstration of loss of a series of stabilizing inter- and intramolecular bonds. The strong correlation between predicted and experimental results support the reported refinement in the three-dimensional structure of the CCK-occupied receptor.