Target recognition of apocalmodulin by nitric oxide synthase I peptides

An increasing number of proteins are found that are regulated by the Ca2+-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 Ca2+-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 Ca2+-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 Ca2+-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 Ca2+ dependency.