Conformational and molecular modeling studies of sulfated cholecystokinin-15 Craig Giragossian, a Shane Stone, b Anna Maria Papini, c Luis Moroder, d and Dale F. Mierke a,e, * a Department of Chemistry, Division of Biology & Medicine, Brown University, Providence, RI 02912, USA b Department of Chemistry, University of Capetown, Rondebosch 7701, South Africa c Department of Organic Chemistry Ugo Schiff, University of Florence, via G. Capponi 9, I-50121 Florence, Italy d Max-Planck Institute of Biochemistry, 82152 Martinsried, Germany e Department of Molecular Pharmacology, Division of Biology & Medicine, Brown University, Providence, RI 02912, USA Received 1 April 2002 Abstract Conformational features of the C-terminal carboxyamidated pentadecapeptide of CCK ðS 19 HRISDRD½SO 4 –YMGWMDF 33 – NH 2 Þ 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 con- formational 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 photo- affinity 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. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Cholecystokinin; CCK; CCK-15; G-protein coupled receptor; NMR solution structure The multimeric family of carboxyamidated cholecy- stokinin peptides (e.g., CCK-58, CCK-33, CCK-22, and CCK-8) displays a high affinity for two pharmacologi- cally distinct guanine nucleotide-binding regulatory protein coupled receptors (GPCRs), the cholecystokinin type 1 and type 2 receptors (CCK 1 –R and CCK 2 –R). These receptors are responsible for the regulation of multiple physiological processes in the digestive (e.g., gallbladder contractions, pancreatic amylase, pepsino- gen and gastric acid secretions, and mucosal and en- terochromaffin-like cell proliferations in the gastric epithelium) and central nervous (e.g., analgesia, anxiety, memory, and satiety) systems [1–3]. CCK 1 and CCK 2 receptor subtypes are readily distinguished on the basis of their affinity for nonsulfated CCK, gastrin, and sub- type selective agonists and antagonists: the carboxy- amidated C-terminal heptapeptide of CCK, sulfated at Tyr 27 , is the minimal sequence necessary for a full acti- vation of CCK 1 –R, whereas for the CCK 2 –R, only the carboxyamidated C-terminal tetrapeptide is required [1]. Differential affinities of CCK receptor subtypes for endogenous and exogenous ligands, despite the high level of sequence homology and common high affinity for sulfated CCK-8, suggest that the two receptors possess unique ligand-binding sites. Given the difficulty associated with crystallizing integral membrane pro- teins, X-ray structures of GPCRs, with the exception of bovine rhodopsin [4], have remained elusive. Therefore, indirect methods such as site-directed mutagenesis [5– 17], receptor chimera [18–20], and photoaffinity labeling [21–25] have been used to deduce the ligand-binding sites of these receptors. Molecular models, based on the intermolecular contact points generated by these tech- niques, though lacking in their ability to characterize conformations of the structurally diverse extracellular domains, have been used with some success to predict alternate interactions. Biochemical and Biophysical Research Communications 293 (2002) 1053–1059 www.academicpress.com * Corresponding author. Fax: +1-401-863-1595. E-mail address: dale_mierke@brown.edu (D.F. Mierke). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)00334-0