Structural Studies of Calmodulin Activation of Estrogen Receptor Alpha

Abstract

The goal is to determine the mechanism of calcium-dependent activation of estrogen receptor alpha (ERa) by calmodulin (CaM) and to ascertain how oxidative stress and oxidative modifications mediate the interactions between CaM, ERa and antiestrogens. Systemic endocrine/antiestrogen therapy is among the most common treatments for estrogen-dependent breast cancers. ERa is the primary target for antiestrogen therapies, and antiestrogen drugs such as tamoxifen and its metabolites (4-hydroxytamoxifen, endoxifen) bind tightly to ERa and inhibit its ability to activate transcription. Recently, it was demonstrated that CaM is an obligate ERa activator. Interestingly, antiestrogens that bind tightly to ERa also bind tightly to CaM. It has been suggested that therapeutic benefit of antiestrogens for estrogen-dependent breast cancers may derive partially from CaM antagonism. Towards our goal of understanding how CaM activates ERa, we have localized the CaM binding region of ERa and initiated structural studies to determine the structure of the complex of CaM with the ERa CaM binding region. Using NMR and SAXS we find that CaM bound to ERa is somewhat extended structurally compared to high affinity CaM complexes. Circular dichroism and fluorescence studies indicate high affinity between CaM and the CaM binding domain of ERa and that upon binding to CaM the CaM binding region of ERa adopts only partial helical character. Binding of CaM to hydrophobic antiestrogens (tamoxifen, 4-hydroxytamoxifen, endoxifen, raloxifene) is eliminated when methionine residues in CaM are oxidized. However, oxidation does not eliminate binding to ERa. Control experiments with CaM mutants (leucine for methionine) indicate methionine residues are not essential for antiestrogen binding. The results are important for understanding CaM activation of ERa and the link between oxidative stress and antiestrogen resistance development. (Supported by the DOD, the Georgia Cancer Coalition, Bruker AXS and the University of Georgia).