Abstract 2891: Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation.

Estrogen Receptor (ER) belongs to the nuclear receptor super family of ligand-triggered transcription factors. ER is present in about 70% of breast cancers and plays a critical role in the biology of breast carcinoma and improves prognosis. Estrogen binds to ER and mediates a cascade of cellular signaling events. It has been shown that several proteins including calcium binding proteins with EF-hand motifs, which control cellular calcium and calcium mediated signaling pathways, including calmodulin (CaM) could bind to unliganded ER and activate ER. CaM has four EF-hand motifs that change conformation upon binding Ca2+ ions. Ca2+-CaM complex binds to the target proteins and initiates various signaling cascades. Each EF-hand motif contains two alpha helices connected by a 12-residue loop. Both X-ray and NMR studies show that the alpha helices of the EF-hand motifs change their position relative to each other, forming an almost perpendicular conformation. This allows calmodulin to increase its binding affinity for a number of target proteins. CaM forms a compact globular conformation by bending its central helix upon binding its target peptides. S100, a soluble protein that is recently reported to interact with ER has only two EF-hand motifs. S100 protein is overexpressed in several cancers and shares a high degree of sequence homology with calmodulin. Methods: Protein-protein interactions of CaM, S100 with ER alpha were simulated using HADDOCK and HEX. Docking results were analyzed in UCSF Chimera and integrated with data from in vitro proteomics experiments to determine the conformations of ER bound EF-hand protein complexes. Proteomic experiments consisted of chemical crosslinking of the corresponding protein complexes followed by tryptic digestion, analyzing the resulting peptide peaks using FTICR- Mass Spectrometry. Results/Discussion: Using the fold information and contact regions obtained from the Mass spectrometry, 3D structures of ER-EF hand protein complexes were reconstructed. These structures of ER best represent the conformational state sensed by the specific interacting partner. ER-S100 complex is distinct from CaM. The lack of the connective peptidic region between the EF-hand pairs in S100 attributed to less interaction coverage indicative of only inducing a partial agonist-like conformation. We believe that the ensemble of ER-EF hand protein complexes generated by our integrated proteomics-assisted protein interaction profiling will shed light on the lingering issue of hormone independent activation of ER at the molecular level. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) Citation Format: David H. Lee, Bethany K. Asare, Suchitra Singh, Rajendram V. Rajnarayanan. Comparative analyses of human estrogen receptor- EF-hand protein complexes: molecular basis for hormone independent activation. [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 2891. doi:10.1158/1538-7445.AM2013-2891