Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP 4 as YAP inhibitor
2019; Springer Nature; Volume: 39; Issue: 1 Linguagem: Inglês
10.15252/embj.2019102406
ISSN1460-2075
AutoresRebecca Vargas, Vy Duong, Han Han, ALBERT TA, Yuxuan Chen, Shiji Zhao, Bing Yang, Gayoung Seo, Kimberly Chuc, Sunwoo Oh, Amal El Ali, Olga V. Razorenova, Junjie Chen, Ray Luo, Xu Li, Wenqi Wang,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoArticle29 November 2019free access Source DataTransparent process Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor Rebecca E Vargas Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Vy Thuy Duong Department of Chemistry, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Han Han Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Albert Paul Ta Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Yuxuan Chen Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Shiji Zhao Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Bing Yang Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Gayoung Seo Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Kimberly Chuc Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Sunwoo Oh Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Amal El Ali Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Olga V Razorenova Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Junjie Chen Corresponding Author [email protected] orcid.org/0000-0002-1493-2189 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Ray Luo Corresponding Author [email protected] orcid.org/0000-0002-6346-8271 Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA Department of Materials Science and Engineering, University of California, Irvine, Irvine, CA, USA Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Xu Li Corresponding Author [email protected] orcid.org/0000-0002-9401-6382 School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China Search for more papers by this author Wenqi Wang Corresponding Author [email protected] orcid.org/0000-0003-4053-5088 Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Rebecca E Vargas Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Vy Thuy Duong Department of Chemistry, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Han Han Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Albert Paul Ta Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Yuxuan Chen Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Shiji Zhao Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Bing Yang Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Gayoung Seo Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Kimberly Chuc Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Sunwoo Oh Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Amal El Ali Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Olga V Razorenova Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Junjie Chen Corresponding Author [email protected] orcid.org/0000-0002-1493-2189 Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Ray Luo Corresponding Author [email protected] orcid.org/0000-0002-6346-8271 Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA Department of Materials Science and Engineering, University of California, Irvine, Irvine, CA, USA Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Xu Li Corresponding Author [email protected] orcid.org/0000-0002-9401-6382 School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China Search for more papers by this author Wenqi Wang Corresponding Author [email protected] orcid.org/0000-0003-4053-5088 Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA Search for more papers by this author Author Information Rebecca E Vargas1,‡, Vy Thuy Duong2,‡, Han Han1,‡, Albert Paul Ta1, Yuxuan Chen1, Shiji Zhao1, Bing Yang1, Gayoung Seo1, Kimberly Chuc1, Sunwoo Oh1, Amal El Ali3, Olga V Razorenova3, Junjie Chen *,4, Ray Luo *,3,5,6,7, Xu Li *,8 and Wenqi Wang *,1 1Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, USA 2Department of Chemistry, University of California, Irvine, Irvine, CA, USA 3Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA 4Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5Department of Chemical and Biomolecular Engineering, University of California, Irvine, Irvine, CA, USA 6Department of Materials Science and Engineering, University of California, Irvine, Irvine, CA, USA 7Department of Biomedical Engineering, University of California, Irvine, Irvine, CA, USA 8School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China ‡These authors contributed equally to this work *Corresponding author. Tel: +1 713 792 4863; E-mail: [email protected] *Corresponding author. Tel: +1 949 824 9528; E-mail: [email protected] *Corresponding author. Tel: +86 0571 88119529; E-mail: [email protected] *Corresponding author. Tel: +1 949 824 4888; E-mail: [email protected] EMBO J (2020)39:e102406https://doi.org/10.15252/embj.2019102406 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Hippo pathway, which plays a critical role in organ size control and cancer, features numerous WW domain-based protein–protein interactions. However, ~100 WW domains and 2,000 PY motif-containing peptide ligands are found in the human proteome, raising a "WW-PY" binding specificity issue in the Hippo pathway. In this study, we have established the WW domain binding specificity for Hippo pathway components and uncovered a unique amino acid sequence required for it. By using this criterion, we have identified a WW domain-containing protein, STXBP4, as a negative regulator of YAP. Mechanistically, STXBP4 assembles a protein complex comprising α-catenin and a group of Hippo PY motif-containing components/regulators to inhibit YAP, a process that is regulated by actin cytoskeleton tension. Interestingly, STXBP4 is a potential tumor suppressor for human kidney cancer, whose downregulation is correlated with YAP activation in clear cell renal cell carcinoma. Taken together, our study not only elucidates the WW domain binding specificity for the Hippo pathway, but also reveals STXBP4 as a player in actin cytoskeleton tension-mediated Hippo pathway regulation. Synopsis Interactions between the Hippo pathway components are often mediated by WW domain binding to PY motifs. Identification of a conserved WW domain sequence that mediates specific interactions with Hippo pathway components uncovers syntaxin-binding protein 4 (STXBP4) as an inhibitor of YAP activity and a potential tumor suppressor in kidney cancer. A "WW-PY" binding specificity exists within the Hippo pathway protein-protein interaction network. A unique 9-amino acid sequence defines the specificity of WW domain-containing protein interactions with Hippo pathway components. WW domain-containing protein STXBP4 inhibits YAP activity via LATS1-mediated phosphorylation. STXBP4 sequesters YAP in a complex with α-catenin and several PY motif-containing Hippo pathway members. STXBP4 functions as a potential tumor suppressor in clear cell renal cell carcinoma. Introduction Signaling proteins often entail modular domains that facilitate protein–protein interactions to assemble functional protein complexes, control enzymatic activity and regulate protein cellular localization (Cohen et al, 1995; Pawson & Scott, 1997). Importantly, the recognition between domains and their peptide ligands is usually specific, thus allowing the transduction of unique information through signaling cascades (Das & Smith, 2000; Hu et al, 2004). The WW domain is a small protein module that is defined by the presence of two tryptophan (W) residues separated apart by ~ 25 amino acids (Sudol et al, 1995b). WW domain and its cognate proline-rich peptide motif have been identified within various protein complexes widely distributed in plasma membrane, cytoplasm, and nucleus. Failure of their recognition is associated with multiple human diseases including Alzheimer's disease (Mandelkow & Mandelkow, 1998; Liu et al, 2007), Huntington's disease (Faber et al, 1998; Passani et al, 2000), Liddle syndrome (Hansson et al, 1995), Golabi-Ito-Hall syndrome (Lubs et al, 2006; Tapia et al, 2010), muscular dystrophy (Bork & Sudol, 1994; Rentschler et al, 1999; Ervasti, 2007), and cancers (Chang et al, 2007; Salah & Aqeilan, 2011). These facts highlight a crucial role of the WW domain-mediated protein–protein interaction in biological processes and tissue homeostasis. WW domain was initially uncovered by characterizing the protein sequence of YAP, a key transcriptional co-activator downstream of the Hippo pathway (Sudol et al, 1995a; Pan, 2010; Jiang et al, 2015). The Hippo pathway is a highly conserved signaling pathway involved in tissue homeostasis, organ size control, and cancer development (Pan, 2010; Halder & Johnson, 2011; Piccolo et al, 2014; Jiang et al, 2015; Yu et al, 2015). In mammals, the Hippo pathway is composed of a kinase cascade (two serine/threonine kinases, MST and LATS; and the adaptors SAV1 for MST and MOB1 for LATS), downstream effectors (YAP and TAZ), and nuclear transcriptional factors (TEADs). MST phosphorylates and activates LATS, which in turn phosphorylates YAP and TAZ. The phosphorylated YAP/TAZ can be recognized by 14-3-3 proteins, retained in the cytoplasm, and eventually targeted by β-TRCP E3 ligase complex for degradation. When the Hippo pathway is inactivated, unphosphorylated YAP/TAZ enter into the nucleus, where they associate with TEAD transcriptional factors to promote the transcription of genes that are involved in proliferation and survival. Notably, many Hippo pathway components and regulators contain either the WW domain or its proline-rich peptide ligand, mostly "PPxY" motif (P, proline; Y, tyrosine; x, any amino acid; hereafter named as "PY" motif) (Sudol, 2010; Salah & Aqeilan, 2011). YAP, TAZ, SAV1, and KIBRA, an upstream component of the Hippo kinase cascade (Yu et al, 2010), are four known WW domain-containing components of the Hippo pathway (Salah & Aqeilan, 2011). In the nucleus, the WW domain of YAP/TAZ is a requirement for their association with a group of nuclear transcriptional factors and regulators that contain the PY motif to regulate gene transcription (Strano et al, 2001, 2005; Ferrigno et al, 2002; Zhang et al, 2011; Haskins et al, 2014; Qiao et al, 2016; Chang et al, 2018; Liu et al, 2018). In the cytoplasm, the PY motif of LATS1/2 is involved in the LATS1/2-mediated YAP/TAZ phosphorylation (Hao et al, 2008; Verma et al, 2018); several PY motif-containing proteins can physically bind the WW domain of YAP/TAZ and promote YAP/TAZ's cytoplasmic translocation (Espanel & Sudol, 2001; Chan et al, 2011; Wang et al, 2011, 2012b, 2014; Zhao et al, 2011; Liu et al, 2013; Michaloglou et al, 2013; Tavana et al, 2016). Moreover, the phosphorylated YAP/TAZ can negatively regulate Wnt pathway by forming a complex with DVL2, which is mediated by the WW domain of YAP/TAZ and the PY motif of DVL2 (Varelas et al, 2010). As a Hippo upstream component, KIBRA can similarly associate with several Hippo PY motif-containing proteins and negatively regulate YAP (Wilson et al, 2014; Tavana et al, 2016). On the other hand, several WW domain-containing proteins have been shown to modulate the Hippo pathway activity by regulating the Hippo PY motif-containing components and regulators (Salah et al, 2011, 2013; Ulbricht et al, 2013; Yeung et al, 2013; Abu-Odeh et al, 2014; Wang et al, 2015). Collectively, these facts suggest that the WW domain and PY motif-mediated protein–protein interaction plays a fundamental role in building up the major framework of the Hippo pathway. Actually, ~ 100 WW domains and 2,000 PY motif-containing peptides have been predicted in the human proteome (Tapia et al, 2010), raising an issue of binding specificity for the proteins containing WW domain and PY motif. Indeed, a large scale of WW domain array screen only confirmed 10% of the tested WW domain–ligand interactions (Hu et al, 2004). Several large-scale proteomic studies exclusively identified a group of PY motif-containing proteins (e.g., LATS1/2, AMOTs, PTPN14) as the binding partners for the Hippo WW domain-containing components (Couzens et al, 2013; Hauri et al, 2013; Wang et al, 2014). These facts indicate the binding specificity for the Hippo WW domain-mediated protein–protein interaction, while the underlying mechanism is still largely unknown. In this study, we demonstrated the WW domain binding specificity for the Hippo pathway proteins and uncovered a highly conserved amino acid sequence required for it. By using this criterion, we identified STXBP4 as a novel Hippo pathway regulator in human proteome. Mechanistically, STXBP4 assembled a complex with α-catenin and several Hippo PY motif-containing components/regulators to negatively regulate YAP when actin cytoskeleton tension is low. Moreover, both TCGA data and tissue array studies suggested STXBP4 as a potential tumor suppressor in human kidney cancer, whose downregulation is significantly correlated with YAP activation in clear cell renal cell carcinoma. Collectively, our study not only elucidated the WW domain binding specificity for the Hippo pathway protein–protein interaction network, but also identified STXBP4 as a Hippo pathway regulator and a potential tumor suppressor in kidney cancer development. Results Binding specificity exists for the Hippo WW domain-containing components We re-analyzed our previously published proteomic data (Wang et al, 2014) for four Hippo WW domain-containing components YAP, TAZ, SAV1, and KIBRA (Fig 1A) and found that most of the known Hippo PY motif-containing proteins (e.g., AMOT, AMOTL1, AMOTL2, LATS1, LATS2, PTPN14, PTPN21, WBP2) were hardly detected in the SAV1-associated protein complex (Fig 1B). Moreover, proteomic analysis of the WW domains isolated from these four Hippo components (Fig EV1A) further confirmed this finding, where the WW domain of YAP, TAZ, and KIBRA, but not that of SAV1, retrieved most of these known Hippo PY motif-containing proteins (Fig 1B). These data suggest that the WW domain of SAV1 is different from that of YAP, TAZ, and KIBRA in associating with the known Hippo PY motif-containing proteins. Figure 1. The Hippo WW domain shows binding specificity with the known Hippo PY motif-containing proteins. (This figure is related to Fig EV1 and Tables EV1, EV2, EV3 and EV4) Schematic illustration of the human Hippo pathway, where the Hippo WW domain-containing components are highlighted. A summary map of cytoscape-generated merged interaction network for the Hippo WW domain-containing components and their WW domains. The Hippo WW domain-containing proteins show binding specificity to the known Hippo PY motif-containing proteins. TAP-MS analysis of a series of WW domain-containing proteins were performed, and their binding with the indicated Hippo PY motif-containing proteins was summarized in a heatmap. The HCIPs for the Hippo WW domain-containing proteins were involved in different signaling pathways compared to those retrieved from the control WW domain-containing proteins. Gene Ontology analysis was performed. Validation of the binding specificity for the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pull-down assay. Validation of the binding specificity for the derived WW domains from the Hippo WW domain-containing proteins. HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pull-down assay. Source data are available online for this figure. Source Data for Figure 1 [embj2019102406-sup-0010-SDataFig1.jpg] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Proteomic analysis of the WW-containing proteins. (This figure is related to Fig 1) Schematic illustration of the Hippo WW domain-containing components. The total spectral counts (TSCs) and corresponding numbers of HCIPs for the indicated proteomic experiments were listed. The overlapped HCIP rate was compared for the full-length protein vs. its WW domain, and Hippo WW domains vs. control WW domains, respectively. Download figure Download PowerPoint Next, we expanded our proteomic analysis for additional 22 WW domain-containing proteins (Fig EV1B; Tables EV1–EV3) and examined their ability to isolate these known Hippo PY motif-containing proteins. Consistent with previous reports (Salah et al, 2013; Ulbricht et al, 2013; Yeung et al, 2013; Abu-Odeh et al, 2014; Wang et al, 2015), WWOX, BAG3, and members of the HECT family of E3 ligases NEDD4L, WWP1, and WWP2 were found to form complexes with the Hippo PY motif-containing proteins such as AMOT family proteins, CCDC85C, and WBP2 (Fig 1C). However, we failed to identify these Hippo PY motif-containing proteins as the binding proteins for other tested WW domain-containing proteins (Fig 1C). Moreover, the high-confident interacting proteins (HCIPs) of the Hippo WW domain-containing components were involved in different signaling pathways from those of the control WW domain-containing proteins (Fig 1D and Table EV4). We also performed proteomic analysis for the WW domains isolated from 13 randomly selected WW domain-containing proteins and found that only 10.2% of the HCIPs were shared by the Hippo and control WW domains (Fig EV1C). Taken together, these results indicate that the WW domains of the Hippo pathway components YAP, TAZ, and KIBRA possess a binding specificity with the known Hippo PY motif-containing proteins. Validation of the Hippo WW domain binding specificity To validate our proteomic findings, we examined the interaction between a series of WW domain-containing proteins and AMOT family proteins. Unlike YAP, TAZ, and KIBRA, SAV1 failed to bind AMOT and AMOTL1 (Fig 1E). Consistently, we hardly detected the association between SAV1 and LATS1 in our experimental setting (Appendix Fig S1A). Moreover, BAG3, WWOX, and several members of the HECT family of E3 ligases can interact with AMOT proteins (Fig 1E), which is consistent with our proteomic study (Fig 1C). However, other tested WW domain-containing proteins as well as their derived WW domains failed to bind AMOT family proteins (Fig 1E and F). These results demonstrate the WW domain binding specificity for the Hippo pathway proteins. A highly conserved amino acid sequence is required for the Hippo WW domain binding specificity To further explore the underlying mechanism, we analyzed the WW domain protein sequence for the Hippo pathway components as well as WWOX, BAG3, and several members of the HECT family of E3 ligases, which can bind the known Hippo PY motif-containing proteins (Fig 2A). Interestingly, in addition to the two tryptophan residues, additional nine amino acids were found to be highly conserved among these WW domains (Fig 2A). We hypothesized that this conserved 9-amino acid sequence could be required for the specific association with the known Hippo PY motif-containing proteins. Figure 2. Identification of a conserved 9-amino acid sequence that determines the Hippo WW domain binding specificity. (This Figure is related to Figs EV2 and EV3; Appendix Figs S1–S3; Table EV5) A. Sequence alignment of the WW domains derived from the WW domain-containing proteins that are known to bind the Hippo PY motif-containing proteins. The two conserved tryptophan restudies were highlighted in purple. Additional conserved amino acid residues were highlighted in yellow. B. Summary of the residue difference in the identified 9-amino acid sequence for the control WW domains. The conserved two tryptophan residues are labeled in gray; the changed residues are labeled in orange; and the unchanged residues are labeled in white. C–G. Validation of the identified 9-amino acid sequence in determining the Hippo WW domain binding specificity. The requirement of the identified 9-amino acid sequence for AMOT association was, respectively, examined for TAZ (C), TAZ-WW domain (D), KIBRA (E), YAP (F) and SAV1 (G). HEK293T cells were transfected with the indicated SFB-tagged constructs and subjected to the pull-down assay. Source data are available online for this figure. Source Data for Figure 2 [embj2019102406-sup-0011-SDataFig2.jpg] Download figure Download PowerPoint To test this hypothesis, we examined the identified 9-amino acid sequence in the control WW domain-containing proteins that failed to bind the Hippo PY motif-containing proteins (Fig 1C) and found that their WW domains have at least one of these nine amino acids replaced by other residues (Figs 2B and EV2A). As for SAV1, the conserved glutamate residue within this 9-amino acid sequence was found changed to a serine in its WW domain (Fig 2A). Consistently, mutating either of these identified nine amino acids to alanine dramatically disrupted the association of AMOT with TAZ (Fig 2C) or its WW domain (Fig 2D). Similar findings were also observed for both KIBRA (Fig 2E) and YAP (Fig 2F). Notably, mutations of the G and E residues among these identified nine amino acids are less detrimental to the Hippo WW-PY interaction as compared with other identified sites (Fig 2C–E). We also tested the conservative substitution for the "E/D", "Y/F", or "F/Y" of this conserved amino acid sequence and found that the association of AMOT with TAZ and KIBRA was not affected by these substitutions (Appendix Fig S1B). Interestingly, an interaction between SAV1 and AMOT was recovered when the unmatched serine residue was replaced by glutamate, allowing SAV1 WW domain to fit the 9-amino acid sequence criterion (Fig 2G). Taken together, these results demonstrate that the identified 9-amino acid sequence determines the WW domain binding specificity for the Hippo pathway proteins. Click here to expand this figure. Figure EV2. Analyses of the identified 9-amino acid sequence in both control WW domains and evolution. (This figure is related to Fig 2; Appendix Figs S1 and S2; Table EV5) Sequence alignment of the WW domains derived from the control WW domain-containing proteins that cannot bind the Hippo PY motif-containing proteins. The two conserved tryptophan restudies were highlighted in purple, and the identified 9-amino acid residues were highlighted in yellow. Evolutionary analysis of the YAP-WW domains. The identified 9-amino acid sequence is highlighted in the two YAP-WW domains derived from the indicated species. A PY motif is identified in Capsapsora owczarzaki LATS. Schematic illustration of the C. owczarzaki LATS protein, where the PY motif is indicated. MBD, MOB1-binding domain. The PY motif is underlined in the C. owczarzaki LATS protein sequence, where the auto-phosphorylation site (S586) and the phosphorylation site (T750) in the hydrophobic motif were shown in red. Download figure Download PowerPoint We also examined the Hippo WW domain-containing components in Drosophila and found that this 9-amino acid sequence was highly conserved in the WW domain of Yorkie and Kibra, while Salvador similarly contains a replacement of the conserved glutamate residue by alanine (Appendix Fig S2). By taking YAP as an example, conservation of this 9-amino acid sequence in the YAP-WW domains can be even tracked to Capsapsora owczarzaki (Fig EV2B and Table EV5), an unicellular specie that is known to contain the functional Hippo pathway components (Sebe-Pedros et al, 2012). Interestingly, in C. owczarzaki, a PY motif was also identified in LATS (Fig EV2C), suggesting that this conserved 9-amino acid sequence may play a crucial role for the Hippo pathway at its premetazoan origin. Role of the 9-amino acid sequence in assembly of a specific WW-PY complex involving the Hippo pathway proteins Next, we analyzed a NMR solution structure of the YAP-WW1 domain (the first WW domain of YAP) and SMAD7-PY motif-containing peptide complex (Aragon et al, 2012). Interestingly, the identified 9 amino acids form as two functional groups. First, together with the second tryptophan (W199 of YAP-WW1), the conserved residues E178, Y188, H192, and T197 were involved in the binding interface with the SMAD7-PY motif (Fig EV3A). Specifically, hydrogen bond (H-bond) formation was, respectively, paired between H192 (YAP-WW1 domain) and Y211 (SMAD7-PY motif), and T197 (YAP-WW1 domain) and P209 (SMAD7-PY motif) (Fig EV3B and C). Hydrophobic contact not only existed within the intramolecular interaction between the W199 and Y188 residues of YAP1-WW domain, but also mediated their intermolecular interaction with the P208 and P209 residues within SMAD7-PY motif, respectively (Fig EV3B and C). E178 (YAP-WW1 domain) functioned in sustaining the intermolecular contact between H192 (YAP-WW1 domain) and Y211 (SMAD7-PY motif) by forming both electrostatic and H-bonding interactions with H192 (Fig EV3B and C). Click here to expand this figure. Figure EV3. Structural analysis of the identified 9-amino acid sequence. (This figure is related to Fig 2 and Appendix Fig S3) Illustration of the identified 9-amino acid residues in the average YAP-WW1/SMAD7-PY structure. The initial structure was derived from NMR solution structure (2LTW). SMAD7-PY peptide was adjusted to 50% transparence to show the residue details on the binding interface. Four contact regions within the YAP-WW1/SMAD7-PY complex were shown in details from the representative top cluster structures. Residues from SMAD7-PY motif peptide were labeled in purple. Hydrogen bond is indicated in blue line. The binding type and the corresponding frequency rate were shown for the indicated inter- and intramolecular residue pairs. Simulation analysis of apo YAP-WW1 domain and its indicated mutants. RMSD value for each mutant simulation (referenced against the average apo YAP-WW1 domain) was shown. Average structures of the indicated YAP-WW1 mutant/SMAD7-PY complexes. The average distance between SMAD7-PY motif peptide and the indicated WW domains was summarized in a table. Download figure Download PowerPoint Second, together with the first tryptophan (W177 of YAP-WW1 domain), the rest residues L173, P174, G176, F189, and P202 formed a hydrophobic cluster at the backside of the YAP-WW1/SMAD7-PY complex (Fig EV3A and C). Although not directly interacted with SMAD7-PY motif, this hydrophobic cluster may maintain a unique YAP-WW1 domain structure to facilitate its binding with SMAD7-PY motif. Since these hydrophobic cluster residues are also frequently replaced by other amino acids in the non-Hippo WW domains (Figs 2B and EV2A), we consider them as part of the determinants for the specific Hippo WW-PY recognition. To further determine the role of this identified 9-amino acid sequence from a structure-based perspective, we mutated each of these conserved residues into alanine in silico and performed root-mean-square deviation (RMSD) analyses using the average unbound (apo) structure of YAP-WW1 domain as a reference. Interestingly, mutating either of the identified residues within the backside
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