Interaction of calmodulin-binding domain peptides of nitric oxide synthase with membrane phospholipids: regulation by protein phosphorylation and Ca(2+)-calmodulin.
Endothelial nitric oxide synthase (eNOS) is unique among the NO synthase isozymes in being modified with myristoyl group, which appears to be necessary for its membrane association. However, the presence of myristoylated eNOS in cytosolic fraction after the stimulation-dependent translocation of the enzyme from membrane to cytosol suggests that other regions may be involved in the eNOS-membrane interaction and its regulation. In this study, we have synthesized a 20-amino acid peptide corresponding to the putative calmodulin-binding domain of human eNOS and studied the interaction of the peptide with calmodulin and with various membrane phospholipids. The peptide formed a stoichiometric complex with calmodulin. Upon addition of various acidic phospholipids, the peptide showed a drastic conformational change from random coil to alpha-helix, as was evidenced by circular dichroism spectroscopy. These results suggest that the same domain of eNOS binds both calmodulin and membrane phospholipids. Furthermore, we found that the synthetic peptide was phosphorylated in vitro by protein kinase C. Phosphorylation of the peptide decreased its interaction with membrane phospholipids. Thus, our results raise the possibility that the calmodulin-binding domain is directly involved in the membrane association of eNOS and that phosphorylation of the domain and Ca(2+)-calmodulin may regulate the interaction. Synthetic peptides corresponding to the calmodulin-binding domains of macrophage and neuronal isozymes showed similar abilities to bind phospholipids, suggesting that the calmodulin-binding domains of NO synthase serve as the phospholipid-binding domains as well.
Matsubara
M
M
Division of Biomedical Polymer Science, Fujita Health University, Aichi, Japan.
Titani
K
K
Taniguchi
H
H
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004727:Endothelium / Q000201:enzymology; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism*; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism
D002148:Calmodulin-Binding Proteins; D008563:Membrane Lipids; D010743:Phospholipids; D019001:Nitric Oxide Synthase; D011493:Protein Kinase C; D002118:Calcium