PNNL

Abstract

Oxidation of Met144/145 in calmodulin (CaM) functions as a sensor of oxidative stress, functioning to induce the nonproductive association of oxidized CaM (CaMox) with the plasma membrane Ca-ATPase (PMCA) and other target proteins to down-regulate cellular metabolism. To understand the mechanism underlying the stabilization of the PMCA in the inhibited state, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of Met144 and Met145 and used circular dichroism (CD) and NMR spectroscopy to identify changes in the molecular interactions between CaM and the CaM-binding sequence of the PMCA that are modulated by methionine oxidation. Neither site-directed mutagenesis nor oxidation of Met144 and Met145 results in large changes in the CD spectra or the positions of cross-peaks in the NMR spectra for calcium-activated CaM, indicating that there are no gross structural changes that substantially alter the backbone fold. Nevertheless, there are significant tertiary structural changes and greater conformational heterogeneity that are apparent in the heteronuclear NMR spectra, where the positions and line-widths of selected residues are shifted and broadened. Similar localized structural changes are apparent upon association with the CaM-binding sequence C28W; however, upon complex formation the heteronuclear NMR spectra indicate a homogeneous binding of CaMox to C28W. Oxidation results in a general trend towards a random coil for residues in the C-terminal helix, as indicated by the changes in the 1H?, 13C?, and 13CO, with a loss of helicity for M144 and M145. Both main chain and side chain methyl group chemical shift changes indicate that, in the complex with C28W, the largest structural changes nclude residues that form the C-terminal hydrophobic pocket and that comprise the intermolecular interface with C28W. Smaller changes in the central linker region and N-terminal domain were also observed, and in the N-terminal domain involved residues that contact the C28W peptide. Using 13C-filtered, 13C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W were observed upon oxidation of M144 and M145, with essentially a complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity in the immediate vicinity of M144 and M145, which results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the CaM binding domain of the PMCA.