Circular dichroism and 1H NMR studies on the structures of peptides derived from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and in complex with calmodulin. Nascent alpha-helical structures are stabilized by calmodulin both in the presence and absence of Ca2+.
There exist two types of nitric-oxide synthase (NOS); constitutive isozymes that are activated by binding calmodulin in response to elevated Ca2+ and an inducible isozyme that binds calmodulin regardless of Ca2+. To study the structural basis of the difference in Ca2+ sensitivity, we have designed synthetic peptides of minimal lengths derived from the calmodulin-binding domain of endothelial NOS (eNOS) and that of macrophage NOS (iNOS). A peptide, KRREIPLKVLVKAVLFACMLMRK, derived from human iNOS sequence, retained the ability to bind to calmodulin both in the presence and absence of Ca2+, while a peptide derived from human eNOS sequence, RKKTFKEVANAVKISASLMG, bound to calmodulin only in the presence of Ca2+. Circular dichroism and two-dimensional 1H nuclear magnetic resonance studies suggested that both peptides assume nascent alpha-helical structures in aqueous solution. When mixed with calmodulin, both peptides showed circular dichroism spectra characteristic for alpha-helix. In contrast to other target proteins, the addition of iNOS peptide to calmodulin did not affect the Ca2+ binding of calmodulin appreciably. The peptide derived from the calmodulin-binding domain of iNOS, therefore, binds in alpha-helical structures both to Ca2+-calmodulin and apo-calmodulin, which is unique among various target proteins of calmodulin.
Matsubara
M
M
Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-11, Japan.
Hayashi
N
N
Titani
K
K
Taniguchi
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004533:Egtazic Acid / Q000031:analogs & derivatives; D004730:Endothelium, Vascular / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D017186:Titrimetry
C035684:5-dimethylaminonaphthalene-1-sulfonyl-calmodulin; D002147:Calmodulin; D007527:Isoenzymes; D010446:Peptide Fragments; C053459:5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004533:Egtazic Acid; D019001:Nitric Oxide Synthase; D002118:Calcium