A Potential Structural Switch for Regulating DNA-Binding by TEAD Transcription Factors

Abstract

TEA domain (TEAD) transcription factors are essential for the normal development of eukaryotes and are the downstream effectors of the Hippo tumor suppressor pathway. Whereas our earlier work established the three-dimensional structure of the highly conserved DNA-binding domain using solution NMR spectroscopy, the structural basis for regulating the DNA-binding activity remains unknown. Here, we present the X-ray crystallographic structure and activity of a TEAD mutant containing a truncated L1 loop, ΔL1 TEAD DBD. Unexpectedly, the three-dimensional structure of the ΔL1 TEAD DBD reveals a helix-swapped homodimer wherein helix 1 is swapped between monomers. Furthermore, each three-helix bundle in the domain-swapped dimer is a structural homolog of MYB-like domains. Our investigations of the DNA-binding activity reveal that although the formation of the three-helix bundle by the ΔL1 TEAD DBD is sufficient for binding to an isolated M-CAT-like DNA element, multimeric forms are deficient for cooperative binding to tandemly duplicated elements, indicating that the L1 loop contributes to the DNA-binding activity of TEAD. These results suggest that switching between monomeric and domain-swapped forms may regulate DNA selectivity of TEAD proteins.

Keywords: Hippo pathway; TEAD; X-ray crystallography; domain swapping; transcription factor.

Figure 1

The TEAD transcription factor. (A) Amino acid sequence of the DNA-binding domain of the human TEAD1 transcription enhancer factor. Amino acids deleted in the ΔL1 TEAD DBD are shown in red. N- and C-terminal amino acids resulting from cloning are in smaller font. Numbering below the sequence, in blue, correspond to the NMR structure. Black numbering and yellow cylinders correspond to the X-ray crystallographic structure (this work, PDB id: 4Z8E). (B) The corresponding three dimensional solution NMR structure of TEAD DBD (PDB id: 2HZD). The L1 loop is shown in red.

Figure 2

DNA binding activity of ΔL1 TEAD DBD. The mutant TEAD DBD binds to double stranded 1xGT (A, top left) and 2xGT (C, top right) DNA. Dissociation constants for binding to 1xGT (B, lower left) and 2xGT (D, lower right) were determined using the non-linear curve fitting routine. The fits for binding to one site (B) and two sites (D) are shown as red curves together with the 95% confidence limit (shaded in cyan). Panel D also shows the fraction single site occupancy on the 2xGT DNA (open circles and gray curve). Arrowhead indicates free DNA, ‘s’ refers to band consisting of one protein molecule per molecule of DNA, and ‘d’ refers to two protein molecules per DNA molecule.

Figure 3. Structure of ΔL1 TEAD DBD

A. Helix-swapped dimer (green and yellow cartoons) comprising two ΔL1 TEAD DBD monomers. Trp9 and His67 side chains are shown as stick models. B. Closest structural homolog of ΔL1 TEAD DBD (yellow) is the Radialis MYB domain (cartoon with blue and pink helices; PDB id: 2CJJ). Superimposition of the two structures reveals that the structural homology includes the Trp-His π stacking interaction. (Superimpose was performed using ‘colorbyrmsd’ script in Pymol; blue = lowest RMSD; pink = highest RMSD, gray = not used in superimpose.) C. Stereo image of the ΔL1 TEAD DBD three-helix bundle formed by domain swapping between two chains. Helix 1 from one chain is shown in yellow. Helices 2 and 3, shown in green, are from the second chain. Hydrophobic side chains involved in formation of the three-helix bundle are shown as sticks. D. Inter-chain contacts between L1 loops of the two domain-swapped ΔL1 TEAD DBD monomers in the unit cell. E. The ΔL1 TEAD DBD crystal structure (yellow), shows interaction between the N-terminal arm and DNA recognition helix through the Trp9 and His67 ring stacking interaction (shown as stick models). In contrast, in the TEAD DBD solution structure (blue) the NTA is unstructured and Trp9 is distant from the His67 side chain.

Figure 4. ΔL1 TEAD DBD exists largely in the monomeric form

A. Results of gel filtration chromatography show that the major peak corresponding to ΔL1 TEAD DBD elutes at volumes corresponding to monomers (red triangle). TEAD DBD elutes at 12kD (green square). Proteins used for molecular weight calibration of the Sephacryl S200 XK 16/60 column are shown as filled diamonds. $$\text{Curvefit}\text{parameters:}\mathrm{y}=-0.198\text{ln}(\mathrm{x})+2.6991;{\mathrm{R}}^2=0.9828$$ B. DNA binding by monomeric and multimeric fractions of TEAD DBD and ΔL1 TEAD DBD. Fractions from size exclusion chromatography that correspond to monomeric TEAD DBD (a), monomeric ΔL1 TEAD DBD, multimeric TEAD DBD (c), and multimeric ΔL1 TEAD DBD (d) were used for EMSA. Arrowhead indicates free DNA at 2 fmol/lane. Protein concentrations: lane 1: 0; lanes 2–8 & 9–15: 0.3, 1, 3, 10, 30, 100, 300 nM. C. SDS-PAGE of the four samples used for EMSA and molecular weight markers (M) (Bio-Rad, Kaleidoscope). (kD): Molecular weights of markers in kilodaltons.

Figure 4. ΔL1 TEAD DBD exists largely in the monomeric form

A. Results of gel filtration chromatography show that the major peak corresponding to ΔL1 TEAD DBD elutes at volumes corresponding to monomers (red triangle). TEAD DBD elutes at 12kD (green square). Proteins used for molecular weight calibration of the Sephacryl S200 XK 16/60 column are shown as filled diamonds. $$\text{Curvefit}\text{parameters:}\mathrm{y}=-0.198\text{ln}(\mathrm{x})+2.6991;{\mathrm{R}}^2=0.9828$$ B. DNA binding by monomeric and multimeric fractions of TEAD DBD and ΔL1 TEAD DBD. Fractions from size exclusion chromatography that correspond to monomeric TEAD DBD (a), monomeric ΔL1 TEAD DBD, multimeric TEAD DBD (c), and multimeric ΔL1 TEAD DBD (d) were used for EMSA. Arrowhead indicates free DNA at 2 fmol/lane. Protein concentrations: lane 1: 0; lanes 2–8 & 9–15: 0.3, 1, 3, 10, 30, 100, 300 nM. C. SDS-PAGE of the four samples used for EMSA and molecular weight markers (M) (Bio-Rad, Kaleidoscope). (kD): Molecular weights of markers in kilodaltons.

Figure 5. A model of DNA binding by TEAD DBD and its ΔL1 mutant

Top: The mutant protein can exist in the monomeric and domain-swapped dimeric forms. Bottom: DNA binding models for monomeric and domain-swapped forms: Bottom Left: monomers of the wild type TEAD, with an intact L1 loop (dotted black line), or ΔL1 TEAD DBD bind to the two binding sites on a direct repeat DNA element independently (A and B), or cooperatively to both sites (C). Bottom Right: DNA binding by the domain-swapped dimer. One molecule of the domain-swapped ΔL1 TEAD DBD may bind to either of the two available sites on the direct repeat DNA (D, E). However, cooperative binding by one domain-swapped dimer to both sites is likely to be unfavorable either because the tethered second domain is likely to be out of register with regard to the adjacent binding site or because of the missing L1 mediated contact protein-protein or protein-DNA contacts (E). Cooperative binding of two molecules of oligomeric/domain-swapped form is likely disallowed, presumably due to steric constraints, and not observed experimentally (F).