Mapping of ligand binding sites of the cholecystokinin-B/gastrin receptor with lipo-gastrin peptides and molecular modeling

Abstract

Double‐tailed lipo‐tetragastrin derivatives of increasing fatty acid chain length were used to identify the minimum size of the fatty acid moieties (≥C10) that restricts the access to the CCK‐B/gastrin (CCK: cholecystokinin) receptor via a membrane‐bound pathway. Then dimyristoyl‐mercaptoglycerol/maleoyl‐gastrin adducts of increasing peptide chain length were synthesized to define the minimal peptide size required for receptor binding affinities comparable to those of underivatized gastrin peptides despite anchorage of the lipid tails in the membrane bilayer. The experimental results indicated that most of the little‐gastrin sequence, i.e., 2–17, is needed for optimal interaction of the molecule with the binding cleft of the receptor. From these data experimentally based restraints could be derived for docking of lipo‐gastrin onto a CCK‐B/gastrin receptor model applying molecular dynamics simulations and energy minimizations. In the receptor‐bound state some of the secondary structure elements of gastrin as determined by nmr analysis of gastrin‐peptides in low dielectric constant media are retained. The N‐terminal gastrin portion interacts in a more or less extended conformation with the receptor surface, and upon a sharp kink at the Ala‐Tyr dipeptide portion the C‐terminal pentapeptide amide part inserts deeply into the helix bundle. Besides Arg‐57 on top of helix 1 of the receptor, for which no potential interaction with the ligand could be detected, the other amino acid residues identified by mutagenesis studies as involved in gastrin recognition were found to interact with the C‐terminal portion of gastrin. Even taking into account the strong limitations of such a model system, it represents an interesting tool for rationalizing the experimental results of the extensive structure‐function studies performed previously on gastrin and to delineate more precisely the putative ligand binding site on the extracellular face of the receptor. © 1997 John Wiley & Sons, Inc. Biopoly 41: 799–817, 1997

Molecular Complex of Cholecystokinin-8 and N-Terminus of the Cholecystokinin A Receptor by NMR Spectroscopy

“…Instead, the structure assumed by CCK-8 in the presence of DPC micelles is the one that binds to the N-terminal receptor fragment. This observation is in agreement with the theory of a membrane-bound pathway for the interaction of peptide hormones with their G-protein coupled receptors (51,(59)(60)(61)(62). This hypothesis states that the ligand first interacts with the membrane, inducing the peptide to adopt a specific conformation, and then through a two-dimensional translation, the peptide hormone interacts with the extracellular portion of the receptor.…”

Cell membranes… and how long drugs may exert beneficial pharmacological activity in vivo

“…An elegant example has been provided by the fatty acid-substituted nonapeptide fragments of cholecystokinin (CCK-9) [46,47] (Figure 2A). Provided that their hydrocarbon tail is sufficiently long, the synthetic constructs become permanently stuck in the membrane.…”

On the ‘micro’-pharmacodynamic and pharmacokinetic mechanisms that contribute to long-lasting drug action

“…E) The 'exosite' hypothesis model stipulates that part of the ligand binds very tightly to an 'exosite' that is located at the interface between the lipid's hydrocarbon chains and the receptor. This allows the 'active' part of the ligand to reach its binding site by pivoting between the receptor's TM domains (left side) [49,61,73] or its extracellular loops (for larger peptides, right side) [70,71]. F) The ability of ligand molecules to undergo several binding--unbinding sequences with the same receptor or those nearby before drifting away (a phenomenon referred to as 'rebinding' [6,16,80]) could be favored if they reach their receptor faster via a random 2D route in the membrane than via a 3D random walk in solution [75][76][77].…”

“…The first mode of approach by this hydrophilic region has been elegantly illustrated with fatty acid-substituted nonapeptide fragments of cholecsystokinin (CCK-9) [70,71]. While the initial elongation of the fatty acid decreased the affinity of these constructs for their receptor, the decrease stalled upon further elongation from the moment on that the fatty acid moiety could no longer escape from the membrane.…”