Abstract
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.
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