Abstract 2503: Calmodulin induced conformational changes in the estrogen receptor alpha: implications for proteomics-assisted drug discovery.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Estrogen mediated signaling affects cell proliferation, differentiation and mammary gland function. Estrogen Receptors (hERs) are present in 70% of breast cancers. Consequently, estrogen mediated hER signaling plays a critical role in breast cancer diagnosis, prognosis, and treatment. Estrogen Receptors are ligand-triggered transcription factors. However, in the absence of cognate estrogenic ligand, hERs can be activated by a variety of other extracellular signals. It has been shown that several proteins like calmodulin (CaM) can bind to unliganded hER and induce transactivation. This is supported by the increased expression of CaM in several malignancies including breast cancer. Calmodulin antagonists have been shown to down regulate hERs and significantly inhibit hER transcriptional activity. It has been reported that melatonin, an indolic hormone produced mainly by the pineal gland, acts as a calmodulin antagonist. Melatonin induces conformational changes in the hER-CaM complex, impairs the binding of estrogen-hER-CaM complex to DNA, and consequently prevents hER-dependent transcription. Moreover the mutant hER (K302G, K303G) is unable to bind CaM, and becomes insensitive to melatonin. Therefore mapping the CaM/hERα interface will provide more insight to the mechanism behind hERα unliganded activation. Methods: Human estrogen receptor alpha (hERα) interactome was subjected to zero length crosslinking and tryptic digestion in the presence and absence of Ca-CaM. The resulting peptides peaks were analyzed using MALDI-FTICR Mass Spectrometry and models of the CaM/hERα interface were produced using Haddock, Sybyl, Molecular Operating Environment (MOE) and Chimera. Results/Discussion: Integrated interactome profiling of hERα-CaM interactions in various reaction conditions revealed that hERα residues 288-300 are required. This peptidic region is located between the DNA and Ligand binding domains of hERα, indicating that hormone binding is not required for the CaM interaction. Furthermore, CaM forms 1:1 and 2:1 complexes with hERα. CaM binding triggers hERα/hERα homodimers in a conformation similar to agonist bound hERα dimer consistent with the data that CaM binding to hERα can trigger transcriptional activity in the absence of estrogen. The model generated can be used to further drug discovery by providing a scaffold for the CaM/ hERα interface and a structural basis for the design and development of drugs that target CaM activated hERα conformation. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) and NSF LSAMP Bridge to Doctorate grant (BR). Citation Format: Bethany K. Asare, Rajendram V. Rajnarayanan. Calmodulin induced conformational changes in the estrogen receptor alpha: implications for proteomics-assisted drug discovery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2503. doi:10.1158/1538-7445.AM2013-2503