Structural and functional analysis of the YAP-binding domain of human TEAD2 Wei Tiana, Jianzhong Yub, Diana R. Tomchickc, Duojia Panb,1, and Xuelian Luoa,1 aDepartments of Pharmacology and cBiochemistry, The University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390; and bDepartment of Molecular Biology and Genetics, Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Edited* by Johann Deisenhofer, The University of Texas Southwestern Medical Center, Dallas, TX, and approved March 1, 2010 (received for review January 8, 2010) The Hippo pathway controls organ size and suppresses tumorigen- Expression of mammalian MST2, LATS1, and MOB1 genes in fly esis in metazoans by blocking cell proliferation and promoting functionally rescues the phenotypes of the corresponding Hpo, apoptosis. The TEAD1-4 proteins (which contain a DNA-binding Wts, and Mats mutants (8–12, 16, 21). Similar to the fly pathway, domain but lack an activation domain) interact with YAP (which the effectors of the mammalian Hippo pathway are transcription lacks a DNA-binding domain but contains an activation domain) factors, including YAP (Yes-associated protein), and TEAD, the to form functional heterodimeric transcription factors that activate mammalian homologs of Yki and Sd, respectively (16, 18, 22, 23). proliferative and prosurvival gene expression programs. The Hippo There are four closely related human TEAD proteins, TEAD1-4. pathway inhibits the YAP-TEAD hybrid transcription factors by All TEAD proteins contain an N-terminal DNA-binding TEA phosphorylating and promoting cytoplasmic retention of YAP.Here (TEF-1, TEC1, and AbaA) domain and a C-terminal YAP-bind- we report the crystal structure of the YAP-binding domain (YBD) of ing domain (YBD) (Fig. 1A). YAP has an N-terminal TEAD- human TEAD2. TEAD2 YBD adopts an immunoglobulin-like β-sand- binding domain and a C-terminal transcriptional activation wich fold with two extra helix-turn-helix inserts. NMR studies domain. Through direct physical interaction, TEAD YBD recruits reveal that the TEAD-binding domain of YAP is natively unfolded YAP to promoters of target genes, where it turns on gene expres- and that TEAD binding causes localized conformational changes in sion. When the Hippo pathway is activated, LATS1/2 phosphory- YAP. In vitro binding and in vivo functional assays define an exten- lates YAP on S127 and promotes its association with 14-3-3 and BIOPHYSICS AND sive conserved surface of TEAD2 YBD as the YAP-binding site. cytoplasmic retention, thus inhibiting the activities of the hybrid COMPUTATIONAL BIOLOGY Therefore, our studies suggest that a short segment of YAP adopts transcription factors formed between YAP and TEAD1-4. an extended conformation and forms extensive contacts with a The Hippo pathway has been implicated in human cancers. In rigid surface of TEAD. Targeting a surface-exposed pocket of TEAD particular, several lines of evidence indicate YAP as an oncogene. might be an effective strategy to disrupt the YAP-TEAD interaction The YAP gene is amplified in several human cancers. The YAP and to reduce the oncogenic potential of YAP. protein is frequently overexpressed in human cancers (12, 24–26). Overexpression of YAP in nontransformed human MCF10A cells Hippo pathway ∣ oncogene ∣ crystallography induces the epithelial-mesenchymal transition, a hallmark of tu- morigenic transformation (24). Overexpression of YAP in mouse he Hippo signaling pathway forms a kinase cascade and con- liver causes dramatic liver overgrowth and eventually tumor for- Ttrols organ size by coordinately regulating both cell prolifera- mation (11, 27). The TEAD proteins are major partners of YAP tion and apoptosis (1–7). This pathway is best characterized in the and collaborate with it to regulate the expression of genes that fruit fly, Drosophila. Through genetic screens in Drosophila for promote cell proliferation and inhibit apoptosis. Thus, under- mutants with defects in imaginal disk growth, several tumor sup- standing how TEAD interacts with YAP will provide insights into pressors were identified as the core components of this pathway, how the Hippo pathway regulates the YAP-TEAD transcription including two kinase complexes, the Ste20 family kinase Hippo factors and may ultimately lead to strategies that can disrupt the (Hpo) in complex with the adaptor protein Salvador (Sav) and tumor-promoting YAP-TEAD interactions. the nuclear Dbf2-related (NDR) family kinase Warts (Wts) Toward this goal, we have determined the crystal structure of bound to its activator Mats (8, 9). Upon activation by extracellular the YAP-binding domain of human TEAD2. We have further stimuli or cell-cell contact, the Hpo-Sav complex phosphorylates analyzed the interactions between TEAD and YAP using in vitro and activates the Wts-Mats complex. The activated Wts-Mats binding assays, in vivo luciferase assays, and NMR spectroscopy. complex in turn phosphorylates the transcriptional coactivator Our studies pinpoint a surface-exposed pocket of TEAD YBD Yorki (Yki) on S168 and creates a binding site for 14-3-3 proteins. that is critical for YAP binding. Targeting this pocket chemically Binding of 14-3-3 causes the cytoplasmic sequestration and inac- might be an effective strategy to disrupt the YAP-TEAD interac- tivation of Yki (10–14). When the Hippo pathway is inactivated, tions and to attenuate the function of YAP. Yki is dephosphorylated by an unknown phosphatase and trans- locates into the nucleus. Because Yki does not contain a Results and Discussion DNA-binding domain, it interacts with the transcription factor Structure Determination of the YAP-Binding Domain of TEAD. The Scalloped (Sd) (which contains a sequence-specific DNA-binding structure of the TEA DNA-binding domain of TEAD proteins domain) to form a functional heterodimeric transcription factor has been determined previously (28). The structure of the (15–18). The Yki-Sd hybrid transcription factor activates the transcription of important target genes, such as the cell cycle gene Author contributions: W.T., D.P., and X.L. designed research; W.T., J.Y., and X.L. performed cyclin E, the antiapoptotic gene Diap1, and the microRNA research; D.R.T., D.P., and X.L. analyzed data; and X.L. wrote the paper. Bantam, thereby promoting cell growth and proliferation and The authors declare no conflict of interest. inhibiting apoptosis (19, 20). By restraining the activities of *This Direct Submission article had a prearranged editor. Yki-Sd, the Hippo pathway controls organ size. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, The Hippo pathway is conserved in mammals (1–7). All core www.pdb.org (PDB ID code 3L15). components in fly have human homologs, including MST1/2 1To whom correspondence may be addressed. E-mail: [email protected] or for Hpo, WW45 for Sav, LATS1/2 for Wts, and MOB1 for Mats. [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1000293107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 24, 2021 48 102S127 171 204 230 263 292 488 A hYAP 1 TBD WW WW AD 504 Table 1. Data collection, structure determination, and refinement 40 114 217 Data collection hTEAD2 14TEA YBD 47Crystal SeMet (peak)* B Space group C2 C N B C Energy (eV) 12,655.6 3 B N Resolution range (Å) 50.0–2.00 (2.03–2.00) 8 12 10 8 5 10 3 Unique reflections 34,385 (1,250) 11 6 1 6 A A Multiplicity 4.9 (3.8) 7 9 7 9 Data completeness (%) 96.1 (70.9) 5 12 R † 1 merge (%) 10.1 (71.1) 4 2 4 11 I∕σðIÞ 10.5 (1.8) 2 D 2 D Wilson B value, Å 31.9 C Phase determination C Selenium (8 of 8 possible Anomalous scatterer sites) Figure of merit, 50–2.00 Å 0.36 N C Refinement statistics Resolution range, Å 33.74–2.00 (2.05–2.00) R ∕R 32; 458∕1; 878 1; 571∕88 No. of reflections work free ( ) Data completeness, % 90.6 (60.0) 8 5 Atoms (non-H protein/solvent/ 9 6 other) 3; 277∕160∕18 R 7 work (%) 18.8 (29.2) 1 4 R (%) 24.1 (34.3) 2 free 3 N C rmsd bond length (Å) 0.01 C rmsd bond angle (°) 1.05 2 Mean B value (Å ) (protein/ PDE Arl2 Overlay solvent/other) 47.9∕54.8∕69.8 Ramachandran plot (%) Fig. 1. Structure of human TEAD2217–447.(A) Schematic drawing of the do- (favored/additional/ ‡ main organization for human YAP and TEAD2 proteins. The residue numbers disallowed) 97.9∕2.1∕0.0 for different domain boundaries are labeled. TBD: TEAD-binding domain; Maximum likelihood coordinate AD: transcriptional activation domain; TEA: DNA-binding TEA domain; error 1.75 YBD: YAP-binding domain. (B) Ribbon diagram of TEAD2217–447 in two views. Chain A: 217–221, 240–247, Strands are colored blue, helices are colored magenta, and loops are colored 257–265, 309–324, 447; gray. The N/C termini and secondary structure elements are labeled. (C Left) Chain B: 217–220, Ribbon diagram of the PDEδ-Arl2 complex. The PDEδ molecule is colored in Missing residues 257–262, 309–324, 447. wheat and the Arl2 molecule is colored in green. (C Right ) Superposition of Data for the outermost shell are given in parentheses. PDEδ and TEAD2217–447. All structural figures were generated with PyMOL. *Bijvoet pairs were kept separate for data processing. †R ¼ 100 ∑ ∑ jI − hI ij∕∑ ∑ I h merge h i h;i h h i h;i , where the outer sum ( )isoverthe YBD of TEAD is unknown, however. We have determined the unique reflections and the inner sum (i) is over the set of independent structure of human TEAD2 YBD containing residues 217–447 by observations of each unique reflection.