Structures of the human cholecystokinin receptors bound to agonists and antagonists.

Cholecystokinin receptors, CCKAR and CCKBR, are important neurointestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here, we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR-gastrin in complex with Gi2 and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

Novel benzodiazepine photoaffinity probe stereoselectively labels a site deep within the membrane-spanning domain of the cholecystokinin receptor.

An understanding of the molecular basis of drug action provides opportunities for refinement of drug properties and for development of more potent and selective molecules that act at the same biological target. In this work, we have identified the active enantiomers in racemic mixtures of structurally related benzophenone derivatives of 1,5-benzodiazepines, representing both antagonist and agonist ligands of the type A cholecystokinin receptor. The parent compounds of the 1,5-benzodiazepine CCK receptor photoaffinity ligands were originally prepared in an effort to develop orally active drugs. The enantiomeric compounds reported in this study selectively photoaffinity-labeled the CCK receptor, resulting in the identification of a site of attachment for the photolabile moiety of the antagonist probe deep within the receptor's membrane-spanning region at Leu(88), a residue within transmembrane segment two. In contrast, the agonist probe labeled a region including extracellular loop one and a portion of transmembrane segment three. The antagonist covalent attachment site to the receptor served as a guide in the construction of theoretical three-dimensional molecular models for the antagonist-receptor complex. These models provided a means for visualization of physically plausible ligand-receptor interactions in the context of all currently available biological data that address small molecule interactions with the CCK receptor. Our approach, featuring the use of novel photolabile compounds targeting the membrane-spanning receptor domain to probe the binding site region, introduces powerful tools and a strategy for direct and selective investigation of nonpeptidyl ligand binding to peptide receptors.

Modeled structure of a G-protein-coupled receptor: the cholecystokinin-1 receptor.

The Cholecystokinin-1 receptor (CCK1R) mediates actions of CCK in areas of the central nervous system and of the gut. It is a potential target to treat a number of diseases. As for all G-protein-coupled receptors, docking of ligands into modeled CCK1R binding site should greatly help to understand intrinsic mechanisms of activation. Here, we describe the procedure we used to progressively build a structural model for the CCK1R, to integrated, and on the basis of site-directed mutagenesis data on its binding site. Reliability of the CCK1R model was confirmed by interaction networks that involved conserved and functionally crucial motifs in G-protein-coupled receptors, such as Glu/Asp-Arg-Tyr and Asn-Pro-Xaa-Xaa-Tyr motifs. In addition, the 3-D structure of CCK1R-bound CCK resembled that determined by NMR in a lipid environment. The derived computational model was also used for revealing binding modes of several nonpeptide ligands and for rationalizing ligand structure-activity relationships known from experiments. Our findings indeed support that our "validated CCK1R model" could be used to study the intrinsic mechanism of CCK1R activation and design new ligands.

Intermolecular interactions between peptidic and nonpeptidic agonists and the third extracellular loop of the cholecystokinin 1 receptor.

Intermolecular interactions were determined between a synthetic peptide corresponding to the third extracellular loop and several residues from the adjoining sixth and seventh transmembrane domains of the human cholecystokinin-1 receptor, CCK(1)-R(329-357), and the synthetic agonists Ace-Trp-Lys[NH(epsilon)CONH-o-(MePh)]-Asp-MePhe-NH(2) (GI5269) and the C1 N-isopropyl-N-(4-methoxyphenyl)acetamide derivative of 3-(1H-Indazol-3ylmethyl)-3-methyl-5-pyridin-3-yl-1,5-benzodiazepine (GI0122), using high-resolution nuclear magnetic resonance spectroscopy and computer simulations. Addition of the ligands to CCK(1)-R(329-357) in an aqueous solution of DPC micelles produced a number of intermolecular nuclear Overhauser enhancements (NOEs) to residues in TMs 6 and 7 of the receptor fragment. NOE-restrained molecular models of the GI5269 and GI0122/CCK(1)-R complexes provide evidence for overlapping ligand-binding sites for peptidic and nonpeptidic agonists. The proposed binding modes of GI5269 and GI0122 are supported by the structure-activity relationship of analogues and mutagenesis data for the CCK(1)-R selective antagonist L-364,718.