Characterization of the Ca2+ -dependent and -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 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 beta-sheet conformation. The interaction 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 contrast to the situation in the presence of Ca2+ in which case the peptide acquired an alpha-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 Kd 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.
Yuan
T
T
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Vogel
H J
HJ
Sutherland
C
C
Walsh
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D002942:Circular Dichroism; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000187:drug effects / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D016415:Sequence Alignment
D002147:Calmodulin; D010455:Peptides; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D009219:Myosin-Light-Chain Kinase; D002118:Calcium