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. 2010 Apr 20;107(16):7293-8.
doi: 10.1073/pnas.1000293107. Epub 2010 Apr 5.

Structural and functional analysis of the YAP-binding domain of human TEAD2

Wei Tian  1 , Jianzhong Yu, Diana R Tomchick, Duojia Pan, Xuelian Luo
Affiliations

Structural and functional analysis of the YAP-binding domain of human TEAD2

Wei Tian et al. Proc Natl Acad Sci U S A. .

Abstract

The Hippo pathway controls organ size and suppresses tumorigenesis in metazoans by blocking cell proliferation and promoting apoptosis. The TEAD1-4 proteins (which contain a DNA-binding domain but lack an activation domain) interact with YAP (which lacks a DNA-binding domain but contains an activation domain) to form functional heterodimeric transcription factors that activate proliferative and prosurvival gene expression programs. The Hippo pathway inhibits the YAP-TEAD hybrid transcription factors by phosphorylating and promoting cytoplasmic retention of YAP. Here we report the crystal structure of the YAP-binding domain (YBD) of human TEAD2. TEAD2 YBD adopts an immunoglobulin-like beta-sandwich fold with two extra helix-turn-helix inserts. NMR studies reveal that the TEAD-binding domain of YAP is natively unfolded and that TEAD binding causes localized conformational changes in YAP. In vitro binding and in vivo functional assays define an extensive conserved surface of TEAD2 YBD as the YAP-binding site. Therefore, our studies suggest that a short segment of YAP adopts an extended conformation and forms extensive contacts with a rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD might be an effective strategy to disrupt the YAP-TEAD interaction and to reduce the oncogenic potential of YAP.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure of human TEAD2217–447. (A) Schematic drawing of the domain organization for human YAP and TEAD2 proteins. The residue numbers for different domain boundaries are labeled. TBD: TEAD-binding domain; AD: transcriptional activation domain; TEA: DNA-binding TEA domain; YBD: YAP-binding domain. (B) Ribbon diagram of TEAD2217–447 in two views. Strands are colored blue, helices are colored magenta, and loops are colored gray. The N/C termini and secondary structure elements are labeled. (C Left) Ribbon diagram of the PDEδ-Arl2 complex. The PDEδ molecule is colored in wheat and the Arl2 molecule is colored in green. (C Right ) Superposition of PDEδ and TEAD2217–447. All structural figures were generated with PyMOL.
Fig. 2.
Fig. 2.
Interactions between YAP and TEAD. (A) Superposition of the 15N/1H-HSQC spectra of YAP and the YAP-TEAD complex, whose cross-peaks are in black and red, respectively. The free YAP cross-peaks that disappear upon TEAD binding are indicated by arrows. (B) ITC analysis of the binding between the YAP61–100 peptide and TEAD2217–447. (C) ITC analysis of the binding between YAP2–268 and TEAD2217–447. The dissociation constant (Kd) and other related parameters of binding are indicated.
Fig. 3.
Fig. 3.
A conserved surface on TEAD. (A) Multiple sequence alignment of TEAD YBD from different species. The secondary structural elements of TEAD2 YBD are drawn above the sequences and colored the same as in the ribbon diagram. Residues identical in all sequences are shaded yellow. Surface-exposed residues in human TEAD2 subjected to mutagenesis in this study (see below) are labeled with asterisks. (B Left) Ribbon diagram of TEAD2 YBD with conserved identical residues shown as yellow sticks. (B Right) Molecular surface of TEAD2 YBD in the same orientation. (C) Same as in B but showing the TEAD molecule in a view that is related to the view in B by a 180° rotation along the y axis. Y442 in TEAD2, which corresponds to the conserved tyrosine in TEAD1 mutated in Sveinsson’s choriorentinal atrophy, is labeled in red.
Fig. 4.
Fig. 4.
The YAP-binding surface of TEAD. (A) 35S-labeled TEAD2 WT or its mutants were incubated with beads bound to GST or GST-YAP2–268. After washing, proteins bound to beads were separated on SDS-PAGE and analyzed using a phosphoimager. Twelve percent of each protein used in the assay was included as input for comparison. (B) Binding of different TEAD2 proteins toward YAP tested in A was quantified and normalized against the wild-type TEAD2. (C) Ribbon diagram of TEAD2 YBD with residues that when mutated abolished YAP binding shown as yellow sticks. (D) The electrostatic surface of TEAD2 YBD in the same orientation as in C. The critical YAP-binding pocket is indicated by a dashed circle.
Fig. 5.
Fig. 5.
The YAP-TEAD interaction is required for gene expression. (A) Schematic drawing of the experimental design. The Gal4-TEAD fusion protein recruits YAP to the UAS elements at the promoter of the luciferase reporter and turns on its expression. (B) Two hundred ninety-three cells were transfected with the indicated plasmids along with the Gal4-responsive luciferase reporter. Luciferase activity of Gal4-TEAD2 WT or Gal4-TEAD2 mutants with or without HA-YAP relative to that of Gal4 DBD alone was plotted. Experiments were performed in triplicates. Error bars represent standard deviations. (C) Cell lysates from B were blotted with anti-Gal4 and anti-HA antibodies to show the expression levels of the Gal4-TEAD fusion proteins and HA-YAP, respectively.
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References

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