Oxidatively modified calmodulin binds to the plasma membrane Ca-ATPase in a nonproductive and conformationally disordered complex.
Oxidation of either Met(145) or Met(146) in wheat germ calmodulin (CaM) to methionine sulfoxide prevents the CaM-dependent activation of the plasma membrane (PM) Ca-ATPase (D. Yin, K. Kuczera, and T. C. Squier, 2000, Chem. Res. Toxicol. 13:103-110). To investigate the structural basis for the inhibition of the PM-Ca-ATPase by oxidized CaM (CaM(ox)), we have used circular dichroism (CD) and fluorescence spectroscopy to resolve conformational differences within the complex between CaM and the PM-Ca-ATPase. The similar excited-state lifetime and solvent accessibility of the fluorophore N-1-pyrenyl-maleimide covalently bound to Cys(26) in unoxidized CaM and CaM(ox) indicates that the globular domains within CaM(ox) assume a native-like structure following association with the PM-Ca-ATPase. However, in comparison with oxidized CaM there are increases in the 1) molar ellipticity in the CD spectrum and 2) conformational heterogeneity between the opposing globular domains for CaM(ox) bound to the CaM-binding sequence of the PM-Ca-ATPase. Furthermore, CaM(ox) binds to the PM-Ca-ATPase with high affinity at a distinct, but overlapping, site to that normally occupied by unoxidized CaM. These results suggest that alterations in binding interactions between CaM(ox) and the PM-Ca-ATPase block important structural transitions within the CaM-binding sequence of the PM-Ca-ATPase that are normally associated with enzyme activation.
Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
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D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013552:Swine; D014908:Triticum / Q000378:metabolism
D002147:Calmodulin; D008715:Methionine; D000252:Calcium-Transporting ATPases; D010100:Oxygen