Characterization of the Ca2+‐dependent and ‐independent interactions between calmodulin and its binding domain of inducible nitric oxide synthase

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Abstract
Most interactions of calmodulin (CaM) with its target proteins are Ca2+‐dependent, but a few Ca2+‐independent CaM‐target protein interactions have been identified. One example is the inducible isoform of nitric oxide synthase (iNOS) expressed in macrophages. We describe here the characterization of the Ca2+‐independent interaction between CaM and a synthetic peptide corresponding to the CaM‐binding domain of murine macrophage iNOS using circular dichroism (CD) spectroscopy. The CD spectrum of free iNOS peptide indicated a β‐sheet conformation. The interaction of iNOS peptide with apo‐CaM in the absence of Ca2+ resulted in the peptide acquiring a type II β‐turn structure. This is in contrast to the situation in the presence of Ca2+ in which case the peptide acquired an α‐helical conformation upon interaction with CaM, i.e. similar to the Ca2+‐dependent interactions of CaM with numerous targets such as myosin light chain kinase (MLCK). Consistent with this similar structural change, iNOS peptide inhibited the Ca2+‐CaM‐dependent activation of smooth muscle MLCK by competing with MLCK for binding to Ca2+‐CaM. The K d of Ca2+‐CaM for iNOS peptide was calculated from competition assays to be 0.3 nM. These results indicate that the structure of the CaM‐binding domain of iNOS is quite different when bound to apo‐CaM than Ca2+‐CaM.