Calcium binding and conformational properties of calmodulin complexed with peptides derived from myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP).
The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein kinase C(PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca2+ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca(2+)-CaM signalling and PKC-mediated phosphorylation cascades. We have studies Ca2+ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca2+ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled to the Ca2+ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca2+ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously. NMR analysis of the Ca(2+)-CaM-MRP peptide complex, as well as CD measurements of Ca(2+)-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast to the alpha-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues Lys-75 and Glu-82.
Porumb
T
T
Division of Molecular and Structural Biology, Ontario Cancer Institute, Toronto, Canada.
Crivici
A
A
Blackshear
P J
PJ
Ikura
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D011493:Protein Kinase C; D002118:Calcium