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

FEBS Letters

Sutherland, Cindy²  Yuan, Tao¹  Vogel, Hans J¹  Walsh, Michael P² 

[1] Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, T2N 1N4, Canada;Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4N1, Canada

关键词: Calmodulin;    Nitric oxide synthase;    Myosin light chain kinase;    Circular dichroism;    Ca2+;    CaM;    calmodulin;    CD;    circular dichroism;    MLCK;    myosin light chain kinase;    NOS;    nitric oxide synthase;   

DOI  :  10.1016/S0014-5793(98)00750-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown