Target recognition of apocalmodulin by nitric oxide synthase I peptides.
An increasing number of proteins are found that are regulated by the Ca(2+)-free state of calmodulin, apocalmodulin. Many of these targets harbor a so-called IQ motif within their primary sequence, but several target proteins of apocalmodulin lack this motif. We investigated whether the Ca(2+)-dependent calmodulin-binding site of nitric oxide synthase I could be transformed into a target site of apocalmodulin. Synthetic peptides representing the wild-type amino acid sequence and several peptides carrying mutations were studied by isothermal titration calorimetry and fluorescence spectroscopy. A single amino acid substitution of a negative charge to a positive charge can convert a classical Ca(2+)-dependent binding site of calmodulin into a target site for apocalmodulin. In addition, the introduction of hydrophobic amino acids increases the apparent binding affinity from the micromolar to the nanomolar range. Binding of wild-type and mutant peptides to Ca(2+)-calmodulin was enthalpically driven, and binding to apocalmodulin was entropically driven. Our data indicate that only a few selected amino acid positions in a calmodulin-binding site determine its Ca(2+) dependency.
Censarek
Petra
P
Institut für Biologische Informationsverarbeitung 1, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Beyermann
Michael
M
Koch
Karl-Wilhelm
KW
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry*; D002151:Calorimetry; D006801:Humans; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010446:Peptide Fragments / Q000737:chemistry*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001059:Apoproteins; D002147:Calmodulin; D010446:Peptide Fragments; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat