Structural basis for Ca2+-induced activation and dimerization of estrogen receptor α by calmodulin

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.

FIGURE 1.

ITC of ER(287–305) binding to CaM (A), CaMN (B), and CaMC (C). The molar ratio of titrant added (CaM/ER-α) is plotted on the horizontal axis. The ITC isotherms (bottom panel) were each fit to a one-site model (solid line). Binding parameters are listed in Table 1.

FIGURE 2.

NMR structures of CaM N-lobe (A–C) and C-lobe (D–F) bound to ER(287–305). Superposition of main chain atoms of 10 lowest energy structures is shown (A and D). Ribbon representation of the energy-minimized average main chain structures is shown (B and E). The CaM N-lobe (cyan) is bound to ER(287–305) (orange), and C-lobe (magenta) is bound to ER(287–305) (white). Close-up view of ER-α interaction with exposed side-chain atoms in CaM is shown (C and F). Side-chain atoms of key residues at the interface are shown as sticks. Hydrophobic side-chain atoms in ER-α (Trp²⁹², Leu²⁹⁶, and Ile²⁹⁸) form detailed contacts with each lobe of CaM, and basic side chains in ER-α (Lys²⁹⁹, Lys³⁰², and Lys³⁰³) form salt bridges with Glu¹⁴ (N-lobe) and Glu⁸⁴ (C-lobe) of CaM.

FIGURE 3.

Mutagenesis of CaM-binding site in ER-α reduces binding to CaM. HEK 293H cells were transiently transfected with Myc-tagged constructs of full-length ER-α (WT) or the indicated ER-α mutant constructs. Equal amounts of protein were subjected to SDS-PAGE and Western blotting (A). In addition, equal amounts of protein lysate were incubated with calmodulin-Sepharose (CaM-Sepharose) or GST beads alone in the presence of 1 mm CaCl₂ (B). After pelleting beads, bound proteins were resolved by SDS-PAGE and transferred to PVDF. Blots were probed with anti-Myc antibodies. Data are representative of three independent experimental determinations.

FIGURE 4.

Structural model of CaM bound to dimeric ER-α (residues 287–552). The main chain structure of the CaM/ER(287–552) complex was generated using the structures above in Fig. 2 (ER-α residues 287–305) attached to the dimeric LBD crystal structure (residues 305–552; Protein Data Bank code 1A52) as described in the text. One polypeptide chain of the ER-α dimer (orange) is bound to the CaM N-lobe (CaMN, cyan) and the other ER-α chain (white) is bound to the CaM C-lobe (CaMC, magenta). E2 is bound to the dimeric LBD (green), and Ca²⁺ is bound to CaM (yellow).

FIGURE 5.

Schematic model of ER-α-mediated transcription regulated by estrogen and Ca²⁺. DNA binding domain (DBD), LBD, and CaM are shown in black, gray, and light blue. The CaM-binding site on ER-α is shown by a red helix. At low Ca²⁺ and low estrogen (E2), ER-α dimer is destabilized by apo-LBD (gray circles). High E2 levels stabilize dimerization of LBD (gray squares). At high Ca²⁺ levels, CaM binding induces dimerization of the hinge domain (red helix) that in turn places the DBD (triangle) in contact with tandem repeat sequence in ERE (37). High Ca²⁺ and E2 levels are both needed to maximally activate transcription (green arrow).