Oxidatively modified calmodulin binds to the plasma membrane Ca-ATPase in a nonproductive and conformationally

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Pull-down of Calmodulin-binding Proteins
07:51

Pull-down of Calmodulin-binding Proteins

Published on: January 23, 2012

J Gao1, Y Yao, T C Squier

  • 1Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.

Biophysical Journal
|March 22, 2001
PubMed

Insights

Oxidized calmodulin (CaM) inhibits the plasma membrane calcium ATPase (PM-Ca-ATPase) by altering its binding site and inducing conformational changes. This structural disruption prevents the enzyme

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Calmodulin (CaM) regulates numerous calcium-dependent processes.
  • Oxidation of methionine residues in CaM can impair its function.
  • CaM activates the plasma membrane Ca-ATPase (PM-Ca-ATPase), a key calcium pump.

Purpose of the Study:

  • To investigate the structural basis for inhibition of PM-Ca-ATPase by oxidized CaM (CaM(ox)).
  • To resolve conformational differences in the CaM-PM-Ca-ATPase complex using spectroscopy.

Main Methods:

  • Circular dichroism (CD) spectroscopy to analyze protein structure.
  • Fluorescence spectroscopy to probe protein conformation and environment.
  • Site-specific labeling with N-1-pyrenyl-maleimide.

Main Results:

  • Oxidized CaM (CaM(ox)) binds to the PM-Ca-ATPase at a distinct, overlapping site compared to unoxidized CaM.
  • CaM(ox) induces increased molar ellipticity and conformational heterogeneity in the bound complex.
  • Despite oxidation, CaM(ox) globular domains maintain a native-like structure upon binding.

Conclusions:

  • Oxidized CaM inhibits PM-Ca-ATPase activity through altered binding interactions.
  • These alterations disrupt essential structural transitions in the PM-Ca-ATPase for activation.
  • The findings elucidate the molecular mechanism of CaM inactivation by oxidation.

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