Structure-Function Relationships of M124Q Calmodulin, a Mutant that Mimics Oxidative Insults

Abstract

Calmodulin (CaM) is a calcium-sensitive regulatory protein that interacts with numerous target proteins, including the ryanodine receptor (RyR). The RyR is a calcium channel responsible for releasing Ca2+ from the sarcoplasmic reticulum to induce muscle contraction. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species produced by normal metabolism. It has been shown that the oxidation level of CaM increases with aging, and contributes to muscle degeneration. Oxidized forms of CaM also have a decreased ability to regulate RyR function, thus disrupting the regulation of muscle contraction. The M124Q CaM mutant mimics the effects of a methionine sulfoxide at position 124, a key residue for CaM's interaction with the RyR. Here, we used dipolar electron-electron resonance (DEER) and NMR to characterize the structural dynamics of the M124Q-CaM. DEER analysis of M124Q-CaM suggests that the mutant exhibits open and closed conformations in micromolar Ca2+, but shifts to the more open conformation in nanomolar Ca2+. Differences in the population distributions were also observed for M124Q-CaM in the presence and absence of a RyR1 peptide that forms part of the CaM binding site. Finally, NMR analysis of wild-type and M124Q-CaM also indicated structural shifts due to the mutation and describe how both the wild-type and M124Q CaM respond to changes in Ca2+ concentration. These initial results give important insights on the structural effects of deleterious oxidative processes on the RyR regulation by CaM.