Abstract
The estrogen receptor α (ER-α) regulates expression of target genes implicated in development, metabolism, and breast cancer. Calcium-dependent regulation of ER-α is critical for activating gene expression and is controlled by calmodulin (CaM). Here, we present the NMR structures for the two lobes of CaM each bound to a localized region of ER-α (residues 287-305). A model of the complete CaM·ER-α complex was constructed by combining these two structures with additional data. The two lobes of CaM both compete for binding at the same site on ER-α (residues 292, 296, 299, 302, and 303), which explains why full-length CaM binds two molecules of ER-α in a 1:2 complex and stabilizes ER-α dimerization. Exposed glutamate residues in CaM (Glu(11), Glu(14), Glu(84), and Glu(87)) form salt bridges with key lysine residues in ER-α (Lys(299), Lys(302), and Lys(303)), which are likely to prevent ubiquitination at these sites and inhibit degradation of ER-α. Mutants of ER-α at the CaM-binding site (W292A and K299A) weaken binding to CaM, and I298E/K299D disrupts estrogen-induced transcription. CaM facilitates dimerization of ER-α in the absence of estrogen, and stimulation of ER-α by either Ca(2+) and/or estrogen may serve to regulate transcription in a combinatorial fashion.
Highlights
Estrogen receptor ␣ (ER-␣) function is controlled by calmodulin (CaM)
We performed ITC binding studies on a variety of hinge domain fragments to define a minimal sequence in ER-␣ that exhibits functional binding to CaM (Fig. 1, supplemental Fig. S1, and Table 1)
We present the NMR structures for the two lobes of CaM each bound to the hinge domain region of ER-␣ (Fig. 2), and we propose a model to explain how CaM stabilizes dimerization of ER-␣ (Fig. 4) and activates transcription (Fig. 5)
Summary
Estrogen receptor ␣ (ER-␣) function is controlled by calmodulin (CaM). Results: Both lobes of CaM interact structurally with the same site on ER-␣ (Trp292 and Lys299). With a transcriptional activation function (AF-1), a core DNA binding domain (with two zinc finger motifs), a central hinge region important for receptor dimerization (residues 248 – 314), and a large C-terminal ligand binding domain (residues 317–599). Ca2ϩ-bound CaM (hereafter referred to as CaM) has been shown to bind directly to ER-␣ in the hinge region (residues 298 –317) [4] This Ca2ϩ-induced binding of CaM to ER-␣ has important implications for breast cancer (9 –11). We present the NMR structures for the two lobes of CaM each bound to a functional fragment of ER-␣ (residues 287–305, called ER[287–305]), and we propose a mechanism to illustrate how CaM-induced dimerization of ER-␣ might regulate transcription. CaM bound to ER-␣ sterically blocks access to key lysine residues (Lys299–Lys303) and explains how CaM prevents ubiquitination at these sites implicated in breast cancer [20, 21]
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