High-Affinity and Cooperative Binding of Oxidized Calmodulin by Methionine Sulfoxide Reductase
Methionines play an important role in modulating protein-protein interactions associated with intracellular signaling, and their reversible oxidation to form methionine sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been suggested to couple cellular redox changes to protein function changes through the action of methionine sulfoxide reductases (Msr). Prior measurements indicate the full recovery of target protein activation upon the stereospecific reduction of oxidized CaM by MsrA, where the formation of the S-stereoisomer of Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase. However, the physiological substrates of MsrA remain unclear, as neither the binding specificities nor affinities of protein targets have been measured. To assess the specificity of binding and its possible importance in the maintenance of CaM function, we have measured the kinetics of repair and the binding affinity between oxidized CaM and MsrA. Reduction of Met(O) in fully oxidized CaM by MsrA is sensitive to protein folding, as repair of the intact protein is incomplete, with > 6 Met(O) remaining in each CaM following MsrA reduction. In contrast, following proteolytic digestion, MsrA is able to fully reduce one-half of the oxidized methionines, indicating that Met(O) within folded proteins are not substrates for MsrA repair. Further, in comparison to free Met(O), the turnover number and Km for oxidized CaM (CaMox) are substantially smaller, indicating that the binding interaction retards Msr recycling to reduce steady-state enzyme activity. Mutation of the active site (i.e., C72S) in MsrA permitted equilibrium-binding measurements using both ensemble and single-molecule measurements obtained by fluorescence correlation spectroscopy (FCS). Multiple MsrA bind tightly to CaMox (Kd = 70 +- 10 nM) with an affinity that is three orders of magnitude greater than the Michaelis constant (KM = 71 +- 8 micromolar). These results indicate that MsrA selectively reduces surface-exposed Met(O) within unstructured sequences and suggest that only a small subset of oxidized proteins are substrates for MsrA, which may selectively modulate the function of key signaling proteins as part of an adaptive response to oxidative stress.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 918862
- Report Number(s):
- PNWD-SA-7459; BICHAW; TRN: US200819%%483
- Journal Information:
- Biochemistry, 45(49):14642-14654, Vol. 45, Issue 49; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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