Interaction of calmodulin with its binding domain of rat cerebellar nitric oxide synthase. A multinuclear NMR study.
The intercellular messenger nitric oxide is produced through the action of nitric oxide synthases, a class of enzymes that is regulated by calcium-calmodulin (CaM). In this work, the interaction of CaM with a 23-amino-acid residue synthetic peptide, encompassing the CaM-binding domain of constitutive rat cerebellar nitric oxide synthase (cNOS), was investigated by various NMR methods. Cadmium-113 NMR studies showed that binding of the cNOS peptide increased the affinity of CaM for metal ions and induced interdomain cooperativity in metal ion binding as earlier observed for complexes of CaM with myosin light chain kinase (MLCK) peptides. By using specific isotopically labeled [13C]methyl-Met and selenomethionine-substituted CaM in two-dimensional proton-detected 13C and 77Se NMR studies, we obtained evidence for the involvement of the Met residues of CaM in the binding of the cNOS peptide. These residues form two hydrophobic surface areas on CaM, and they are also involved in the binding of other target proteins. A nitroxide spin-labeled version of the cNOS peptide caused broadening only for NMR resonances in the N-terminal half of CaM, showing that the peptide binds with a C to N orientation to the N- and C-terminal domains of CaM. pH titration experiments of CaM dimethylated with [13C]formaldehyde show that Lys-75 (and Lys-148) experience a large increase in pKa upon peptide binding; this indicates an unraveling of part of the helical linker region of CaM upon cNOS peptide binding. Taken together, our data show that the cNOS and MLCK peptides bind in a closely analogous fashion to CaM.
Zhang
M
M
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Yuan
T
T
Aramini
J M
JM
Vogel
H J
HJ
D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002531:Cerebellum / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000032:analysis; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity
D002147:Calmodulin; D010455:Peptides; D008715:Methionine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases