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

  • Yuan T
  • Vogel H
  • Sutherland C
 et al. 
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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 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

Author-supplied keywords

  • Nitric-Oxide Synthase
  • amino acid sequence
  • animal
  • binding sites
  • calcium
  • calmodulin
  • cattle
  • chemical synthesis
  • chemistry
  • chickens
  • circular dichroism
  • drug effects
  • enzyme activation
  • light
  • metabolism
  • molecular sequence data
  • myosin
  • myosin-light-chain kinase
  • nitric oxide
  • peptides
  • proteins
  • sequence alignment
  • support,non-u.s.gov't

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Authors

  • T Yuan

  • H J Vogel

  • C Sutherland

  • M P Walsh

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