CHARACTERIZATION OF THE CA2(-DEPENDENT AND CA2(+)-INDEPENDENT INTERACTIONS BETWEEN CALMODULIN AND ITS BINDING DOMAIN OF INDUCIBLE NITRIC-OXIDE SYNTHASE())

Most interactions of calmodulin (CaM) with its target proteins are Ca2 +-dependent, but a few Ca2+-independent CaM-target protein interaction s 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 m acrophage iNOS using circular dichroism (CD) spectroscopy. The CD spec trum of free iNOS peptide indicated a beta-sheet conformation. The int eraction of iNOS peptide with apo-CaM in the absence of Ca2+ resulted in the peptide acquiring a type II beta-turn structure. This is in con trast to the situation in the presence of Ca2+ in which case the pepti de acquired an alpha-helical conformation upon interaction with Call, i,e, similar to the Ca2+-dependent interactions of CaM with numerous t argets such as myosin light chain kinase (MLCK). Consistent with this similar structural change, iNOS peptide inhibited the Ca2+ CaM-depende nt 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 stru cture of the CaM-binding domain of iNOS is quite different when bound to apo-CaM than Ca2+-CaM. (C) 1998 Federation of European Biochemical Societies.