The role of segment 32-47 of cholecystokinin receptor type A in CCK8 binding: synthesis, nuclear magnetic resonance, circular dichroism and fluorescence studies.
The segment 32-47 of the N-terminal extracellular domain of the type A cholecystokinn 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 CCKA receptor.
De Luca
Stefania
S
Centro Interuniversitario di Ricerca sul Peptidi Bioattivi & Istituto di Blostrutture e Bioimmagini, CNR, 80134 Napoli, Italy.
Ragone
Raffaele
R
Bracco
Chiara
C
Digilio
Giuseppe
G
Tesauro
Diego
D
Saviano
Michele
M
Pedone
Carlo
C
Morelli
Giancarlo
G
D002942:Circular Dichroism; D005453:Fluorescence; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D043886:Receptor, Cholecystokinin A / Q000737:chemistry* / Q000378:metabolism*; D012844:Sincalide / Q000378:metabolism*; D012997:Solvents; D017186:Titrimetry
D010446:Peptide Fragments; D043886:Receptor, Cholecystokinin A; D012997:Solvents; D012844:Sincalide