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The role of segment 32–47 of cholecystokinin receptor type A in CCK8 binding: synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies
Author(s) -
De Luca Stefania,
Ragone Raffaele,
Bracco Chiara,
Digilio Giuseppe,
Tesauro Diego,
Saviano Michele,
Pedone Carlo,
Morelli Giancarlo
Publication year - 2003
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.442
Subject(s) - micelle , cholecystokinin , chemistry , cholecystokinin receptor , circular dichroism , titration , receptor , binding site , biophysics , ligand (biochemistry) , stereochemistry , crystallography , biochemistry , biology , organic chemistry , aqueous solution
The segment 32–47 of the N ‐terminal extracellular domain of the type A cholecystokinin receptor, CCK A ‐R(32–47), was synthesized and structurally characterized in a membrane mimicking environment by CD, NMR and molecular dynamics calculations. The region of CCK A ‐R(32–47) encompassing residues 39–46 adopted a well‐defined secondary structure in the presence of DPC micelles, whereas the conformation of the N ‐terminal region (segment 32–37) could not be uniquely defined by the NOE derived distance constraints because of local flexibility. The conformation of the binding domain of CCK A ‐R(32–47) was different from that found for the intact N ‐terminal receptor tail, CCK A ‐R(1–47). To assess whether CCK A ‐R(32–47) was still able to bind the nonsulfated cholecystokinin C ‐terminal octapeptide, CCK8, a series of titrations was carried out in SDS and DPC micelles, and the binding interaction was followed by fluorescence spectroscopy. These titrations gave no evidence for complex formation, whereas a high binding affinity was found between CCK A ‐R(1–47) and CCK8. The different affinities for the ligand shown by CCK A ‐R(32–47) and CCK A ‐R(1–47) were paralleled by different interaction modes between the receptor segments and the micelles. The interaction of CCK A ‐R(32–47) with DPC micelles was much weaker than that of CCK A ‐R(1–47), because the former receptor segment lacks proper stabilizing contacts with the micelle surface. In the case of SDS micelles CCK A ‐R(32–47) was found to form non‐micellar adducts with the detergent that prevented the onset of a functionally significant interaction between the receptor segment and the micelle. It is concluded that tertiary structure interactions brought about by the 1–31 segment play a key role in the stabilization of the membrane bound, biologically active conformation of the N ‐terminal extracellular tail of the CCK A receptor. Copyright © 2003 European Peptide Society and John Wiley & Sons, Ltd.