Physical studies of hydrophobic drug binding to calmodulin and the mediation of drug binding by methionine oxidation
Niedermaier, Henry Thomas
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The calcium signal transducer protein calmodulin (CaM) is central in eukaryotic biology. In response to increased intracellular calcium, CaM binds and activates hundreds of cellular proteins. CaM binds to hydrophobic, positively charged regions of target proteins via deep hydrophobic pockets in each of two opposing globular domains rimmed by negatively charged surface patches. CaM also binds to highly hydrophobic small drug and drug-like molecules in its hydrophobic pockets, presumably via primarily hydrophobic mechanisms. The hydrophobic pockets of CaM include nine methionine residues that contact hydrophobic side chains of the target proteins and hydrophobic regions of drug molecules in the respective complexes. In response to conditions of oxidative stress, methionine residues of CaM are readily oxidized to the more polar methionine sulfoxides, thereby increasing the polarity of the hydrophobic pockets of the globular domains of CaM and changing how CaM binds to and activates target proteins. This is important because studies have demonstrated that oxidized CaM can accumulate in cells under conditions of oxidative stress (for instance, aging, cancer), and given the large number of CaM targets in the cell, the consequences to cellular biology could be very significant. Although numerous studies have reported the effects of methionine oxidation in CaM on the interactions of CaM with target proteins, there have been none to date documenting the effects on CaM interactions with small drug and drug-like molecules. My research has explored the binding interactions of CaM with a class of antiestrogen molecules known as selective estrogen receptor modulators (SERMs). It is important to do so because the in-vivo efficacy of these drugs is thought to result in part from CaM inhibition, even though CaM may not be the intended drug target. This dissertation covers my research using NMR spectroscopy and other physical methods to examine drug binding by CaM and how is it altered by oxidation of the methionine side chains in CaM.