Spectroscopic characterization of the calmodulin-binding and autoinhibitory domains of calcium/calmodulin-dependent protein kinase I.
Structural studies of the calmodulin-dependent protein kinase I have shown how the calmodulin-binding domain and autoinhibitory domain interact with the active sites of the enzyme. In this work, we have studied the interaction in solution of two synthetic short and long (22- and 37-residue) peptides representing the binding and autoinhibitory domains of CaMKI with Ca2+-CaM using CD, NMR, and EPR spectroscopy. Both peptides adopt alpha-helical structure when bound to Ca2+-CaM, as detected by CD spectroscopy. Cadmium-113 NMR showed that both peptides induced cooperativity in metal ion binding between the two lobes of the protein. To directly observe the effect of the peptides upon CaM in solution, biosynthetically isotope labeled [methyl-13C-Met]CaM was prepared and studied by 1H, 13C NMR. The relaxation effects of two nitroxide spin-labeled derivatives of the short peptide showed the N-terminal portion of the CaM-binding domain interacting with the C-lobe of CaM, while the C-lobe of the peptide binds to the N-lobe of CaM. Our results are consistent with Trp303 and Met316 acting as the anchoring residues for the C- and N-lobes of CaM, respectively. The NMR spectra of the long peptide showed further differences, suggesting that additional interactions may exist between the autoinhibitory domain and CaM.
Department of Biological Sciences, University of Calgary, 2500 University Drive, N. W., Calgary, Alberta, Canada T2N 1N4.
Tao
T
Yuan
Aldrin V
AV
Gomes
Junor A
JA
Barnes
Howard N
HN
Hunter
Hans J
HJ
Vogel
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002104:Cadmium / Q000378:metabolism; D019703:Calcineurin / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002942:Circular Dichroism; D009682:Magnetic Resonance Spectroscopy; D009589:Nitrogen Oxides / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats
D002147:Calmodulin; D009589:Nitrogen Oxides; D010455:Peptides; D002104:Cadmium; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; C039900:nitroxyl