Crystal structure of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-beta receptor promoter DNA

Abstract

The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-beta receptor gene (TbetaR-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, the A-site and the B-site. Here, we report the 2.2 A resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-beta receptor promoter DNA (mTbetaR-II(DNA)). Elf3 contacts the core GGAA motif of the B-site from a major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity.

Figure 1

Overall view of mElf3_269–371 bound to a B-site of mTβR-II_DNA. (A) mTβR-II_DNA sequences containing A and B Elf3 binding sites. The core regions of A- and B-sites are boxed. The DNA fragment used for crystallization is shown in bold. Highlighted G is replaced by C to enhance the crystallization. (B) The structure of the mElf3_269–371·mTβR-II_DNA complex. The protein is drawn as a ribbon diagram and DNA is represented as a stick diagram. The amino acid residues involved in direct DNA base interactions are also drawn in stick form.

Figure 2

Elf3 domain structure and sequence alignment. (A) Schematic diagram showing the domains of Elf3. (B) Structure-based sequence alignment of the DNA-bound Ets domains of Elf3, PDEF (1yo5), PU.1 (1pue), Sap1 (1k60), Elk1 (1dux), Ets1 (1k79), and GABPα (1awc). The secondary structure elements of Elf3 are indicated above the corresponding sequence. Helices are depicted by red rectangles and β strands as yellow arrows. The α3 helix terminates with a distorted 3₁₀ helix. Elf3 residues missing from the structure are highlighted in grey. Conserved residues are shown in red. The Elf3 residues interacting with DNA are underlined with brown color. Amino acid residues of Ets domains in direct contact with DNA bases are highlighted in cyan. (C) A stereoview of the three-dimensional alignment of Ets domains in the crystal structures of DNA-bound Elf3, PDEF, PU.1, Sap1, Elk1, Ets1, and GABPα. The α-carbon traces are shown as color-coded lines. The α-carbon position of every tenth amino acid residue of Elf3, starting from residue 280, is marked by a small circle.

Figure 3

A summary of the Elf3·mTβR-II_DNA interactions. (A) A schematic representation of protein·DNA interactions. Hydrogen bonds are shown with black lines and van der Waals contacts are shown with magenta lines. Water molecules are represented by red balls. The DNA base pairs in the B-site core region are highlighted in green and DNA base pairs outside the core region that interact with the protein are highlighted in yellow. The Elf3 sequences involved in contacts with DNA bases are highlighted in cyan. (B and C) A stereoview of protein·DNA interface showing the details of Elf3’s direct interactions with (B) DNA bases and (C) DNA backbone. The hydrogen bonds are drawn with black dashed lines and the van der Waals interactions are drawn with dashed magenta lines.

Figure 3

A summary of the Elf3·mTβR-II_DNA interactions. (A) A schematic representation of protein·DNA interactions. Hydrogen bonds are shown with black lines and van der Waals contacts are shown with magenta lines. Water molecules are represented by red balls. The DNA base pairs in the B-site core region are highlighted in green and DNA base pairs outside the core region that interact with the protein are highlighted in yellow. The Elf3 sequences involved in contacts with DNA bases are highlighted in cyan. (B and C) A stereoview of protein·DNA interface showing the details of Elf3’s direct interactions with (B) DNA bases and (C) DNA backbone. The hydrogen bonds are drawn with black dashed lines and the van der Waals interactions are drawn with dashed magenta lines.

Figure 4

Location of Elf3 C-terminal. The structure of mElf3·mTβR-II_DNA is drawn as cartoons. The side-chains of Ets domain hydrophobic core residues and Trp361 are shown as sticks and the side chains of other residues are shown as lines. The Elf3 C-terminal residues 355–366 are in magenta color and the rest of residues are colored by atom type: the carbon, nitrogen and oxygen atoms are in grey, blue and red, respectively.

Figure 5

Binding of Elf3 to the TGFβ-RII EBS B-site in the presence of A-site mutations. EMSA was performed with recombinant Elf3_269–371 and ³²P-labeled probes based on the TGFβR-II EBS sequence. Bands produced by binary and, where present, ternary complexes as well as the free probe are shown. (A) The wild-type probe was incubated with the indicated amount (μM) of Elf3_269–371. (B) The sequence of the wild-type (WT) probe is shown with the core of Ets-binding sites B and A in bold. This sequence is compared to those of mA2, which has two modified base pairs in the A site, and mA4, which has four motified base pairs in the A site. (C) Incubations included 2 μM of Elf3_269–371 and either the WT, mA2 or mA4 probe. As a control, the WT probe was incubated without Elf3 (0). It is evident in part A of this figure that 2 μM of Elf3_269–371 generates little or no ternary complex. Either probe mA2 (D) or mA4 (E) was incubated with 2 μM Elf3_269–371 and 5-fold to 50-fold excess of unlabelled competitor probe mA2 or mA4 was added as indicated. The band produced by the binary complex is shown.

Figure 6

Transcriptional activity of Elf3 and its point mutants. (A) F9-differentiated cells were transiently transfected with the promoter/reporter construct mTGFβ-RII (−108/+56) and 1 or 3 μg of an expression plasmid for flag-tagged Elf3 with either the wild-type (WT) or indicated point mutant sequence. CAT reporter gene activity was measured and normalized as described in Materials and Methods. Independent clones of plasmids for the N357A and W361A mutants gave similar results and this experiment was repeated twice to verify the intermediate effects of the R349A clone. (B) Western blot analysis was performed using cell nuclear extracts from 293T cells transfected with the expression plasmids used in part (A) and an antibody against the N-terminal flag tag to visualize relative expression levels.

Figure 7

Elf3 binding to the TβR-II promoter. (A) A cartoon representation of the model of Elf3 bound to the A- and B-sites of the TβR-II promoter. The DNA fragment shared by both Elf3 molecules is marked by dashed lines (B) A schematic representation of the two-step mechanism of Elf3 binding to the TβR-II promoter. The first Elf3 molecule binds to the high-affinity B-site and induces conformational changes in the A-site (dashed lines). The conformational changes in the A-site allow binding of a second Elf3 molecule to the major groove of the A-site.