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 (CaMox), 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 CaMox indicates that the globular domains within CaMox 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 GD spectrum and 2) conformational heterogeneity between the opposing globular domains for CaMox bound to the CaM-binding sequence of the PM-Ca-ATPase. Furthermore, CaMox 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 CaMox 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.