Structural basis of Ets1 cooperative binding to palindromic sequences on stromelysin-1 promoter DNA

Abstract

Ets1 is a member of the Ets family of transcription factors. Ets1 is autoinhibited and its activation requires heterodimerization with a partner protein or DNA-mediated homodimerization for cooperative DNA binding. In the latter case, Ets1 molecules bind to palindromic sequences in which two Ets-binding sites (EBS) are separated by four base pairs, for example in the promoters of stromelysin-1 and p53. Interestingly, counteraction of autoinhibition requires the autoinhibitory region encoded by exon VII of the gene. The structural basis for the requirement of autoinhibitory sequences for Ets1 binding to palindromic EBS still remains unresolved. Here we report the crystal structure of two Ets1 molecules bound to an EBS palindrome of the stromelysin-1 promoter DNA, providing a plausible explanation for the requirement of exon VII-encoded sequences for Ets1 cooperative DNA binding. The proposed mechanism was verified both in vitro by surface plasmon resonance and in vivo by transcription-based assays.

Figure 1

Overall view of (Ets1)₂•DNA complex. (A) Cartoon representation of (Ets1)₂•DNA complex (top) and the same complex with the Ets1 molecule drawn as surfaces and rotated 180° to highlight DNA docking (bottom). Each interaction area I in the top image is marked by shadow. A close-up view of intermolecular (B) hydrogen bonds and (C) van der Waals contacts in interaction area I. The hydrogen bonds are shown as black dashed lines and the van der Waals interactions are shown as pale green dashed lines.

Figure 2

Intermolecular interactions between two (Ets1)₂•DNA complexes. The molecules are drawn as cartoons with cylindrical α-helices and each Ets1 is shown in different color. The approximate locations of interaction areas II are shadowed in the left image. The right image is a 90° rotation of the left image.

Figure 3

The Ets1•Ets1 interactions in area II. (A) The docking of HI1 helix (yellow ribbon) into the surface of the Ets domain (light orange). The Ets1 fragments involved in intermolecular interactions are highlighted by pale cyan. (B) View as in part (A) except the Ets domain is drawn as cartoon. (C) Comparison of HI1 helix interaction with the Ets domain in crystal structures of (Ets1)₂•DNA and the Ets1 dimer (1gvj). The HI1 and HI2 helices and the Ets domain of the (Ets1)₂•DNA structure are colored in yellow and sand, respectively. The corresponding residues of the Ets1 dimer structure are colored in cyan and blue, respectively. (D) A view of the portion of 2F_o-F_c Fourier map (mesh) contoured at 1σ and covering the residues of the HI1 helix and those in its surroundings.

Figure 4

The model of (Ets1)₂•DNA complex formation. (A) Alignment of amino acid sequences of helix HI1 with the inhibitory sequences N-terminal to HI1. The aligned aromatic residues are highlighted in red, and the serines 282 and 285 which phosphorylation enhances the Ets1 autoinhibition are marked in yellow. The dark red bars above and below the sequences indicate the residues capable of forming helices HI1′ and HI1, respectively. (B) The cartoon of Ets1 cooperative binding to the EBS palindrome. In DNA-free Ets1, the HI1 helix interacts with the Ets domain and directs the N-terminal disordered sequences containing the phosphorylated serines to mask the DNA-binding surface of Ets1. Upon binding to the EBS palindrome, the HI1 dissociates from the Ets domain and becomes disordered. Instead, the HI1′ sequences become folded as a helix which binds to the Ets domain of neighboring Ets1. This enhances the stability of interaction area I (dashed areas). The HI' binding also places phosphoserines (yellow stars) away from the DNA-binding surface of Ets1, reducing their inhibitory function.

Figure 5

In vitro studies of Ets1 cooperative binding to stromelysin-1 promoter. (A) Effects of wild-type Ets1 and Ets1 mutants on the Stromelysin-1 promoter. 293T cells were transfected with 2 µg of the promoter/reporter gene construct plus 50 ng of the TK-RL normalizing vector. (B) Effects of Ets1 on the activity of the Stromelysin-1 promoter with wild-type and disrupted EBS. 293T cells in 60 mm tissue culture dishes were transfected with 2 µg of the promoter/reporter gene constructs pGL3 WT and pGL3 M1M2 plus 50 ng of the TK-RL normalizing vector. In both parts, where indicated, Ets1 expression vectors were added. Reporter activity was normalized to that of TK-RL and the activity of pGL3 WT was set to 1. All other values are shown relative to the promoter in the absence of transfected Ets1 vectors. The data shown represents the mean and standard deviation of duplicate samples from a representative experiment. Each experiment was repeated at least twice and similar results were obtained.

Figure 6

Photomicrograph of (Ets1)₂•DNA crystals. (A) Orthorhombic and (B) monoclinic crystals of (Ets1)₂•DNA.