Recombinant preparation and characterization of interactions for a calmodulin-binding chromogranin A peptide and calmodulin.
Chromogranin-derived peptides have important and varied biological activities. They affect a wide spectrum of targets such as fungal membranes, blood vessels, myocardial cells, and pancreatic cells. Despite the biological significance and the diverse activities, the molecular mechanisms of the interactions between the peptides and the target proteins have not been well understood. Here, we studied the interaction between a chromogranin A-derived peptide (CGA40-65) and its target protein, calmodulin, with NMR spectroscopy. Calmodulin was easily prepared with standard recombinant technology, but CGA40-65 posed challenges requiring multistep procedures. The recombinantly produced peptide retained the calmodulin-binding property of the full-length CGA, as shown by the HSQC binding experiment. By applying resonance assignments, we identified the residues in calmodulin involved in the CGA40-65 binding. We also found that the peak changes are close to those exhibited by the peptides having the wrap-around binding mechanism. Further analysis revealed that the CGA40-65-induced changes are more similar to those by CaMKIp peptide than those by smMLCKp peptide among the wrap-around binding peptides, suggesting that CGA40-65 can be categorized as a CaMKIp-like peptide. Our report is the first residue-resolution mechanistic study involving chromogranin peptides and their target proteins. Our approaches should be applicable to interaction studies involving other chromogranin-derived peptides and their cellular target proteins.
Kang
Sunmi
S
Department of Biochemistry, Center for Advanced Medical Education by BK21 project, School of Medicine, Inha University, Shinheung-dong, Chung-gu, Incheon, Korea.
Kang
Jinho
J
Yoo
Seung Hyun
SH
Park
Sunghyouk
S
D001483:Base Sequence; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053379:Chromogranin A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D053379:Chromogranin A; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins