Neuronal nitric oxide synthase-membrane phospholipid interactions.
Most of the neuronal nitric oxide synthase (nNOS) is present in the particulate fraction of tissue extracts. Here, we show that the calmodulin (CaM)-binding domain of nNOS interacts with anionic phospholipid vesicles but not with neutral ones. Identification of residues in the CaM-binding domain of nNOS as the key domain for the interaction is also documented. Recombinant wild-type nNOS was found to associate with phosphatidylserine (PS) or phosphatidic acid (PA) but not with phosphatidylethanolamine (PE) or phosphatidylcholine (PC), indicating that nNOS-phospholipid binding requires an electrostatic interaction. A synthetic peptide corresponding to residues 732-754 blocked the interaction of nNOS with PS. Furthermore, a purified fusion protein containing residues 724-755 interacted with PS in a competitive fashion with CaM. Inactive nNOS lacking CaM-binding ability, generated by mutation of (Lys732LysLeu) to (Asp732AspGlu) (Watanabe, Y., Hu, Y., and Hidaka, H., FEBS Lett. 403, 75-78, 1997) did not interact with PS. Preincubation of nNOS with PS protected subsequent limited proteolysis of the synthase by Staphylococcus aureus V8 protease, probably as a result of conformational changes in the protein. Wild-type nNOS was found almost entirely in the membrane fraction of Sf9 cells, whereas inactive nNOS was also found in cytosolic fraction in Sf9 cells expressing the mutant enzyme. These results demonstrate that the mutated hydrophobic/basic amino acid cluster in nNOS sequence, Lys732LysLeu, is essential for nNOS-PS and nNOS-CaM interactions.
Watanabe
Y
Y
Department of Pharmacology, Nagoya University School of Medicine, Showa-ku, Nagoya, 466, Japan.
Nishio
M
M
Hamaji
S
S
Hayashi
Y
Y
Hu
Y
Y
Hidaka
H
H
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001921:Brain; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D018411:Spodoptera / Q000378:metabolism
D000596:Amino Acids; D002147:Calmodulin; D008051:Lipid Bilayers; D008081:Liposomes; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase