Article A Tunable Brake for HECT Ubiquitin Ligases Graphical Abstract Authors Zan Chen, Hanjie Jiang, Wei Xu, ..., L. Mario Amzel, Sandra B. Gabelli, Philip A. Cole Correspondence [email protected] (S.B.G.), [email protected] (P.A.C.) In Brief Chen et al. describe an autoinhibitory Phosphorylation mechanism for WWP2, WWP1, ITCH, and NEDD4-1 ubiquitin ligases involving a linker-HECT domain interaction. This intramolecular interaction traps the HECT Autoinhibited HECT Activated HECT enzyme in its inactive state and can be • Blocked allosteric • Moderate activation: relieved by linker phosphorylation. ubiquitin binding site Substrate ubiquitination • Locked hinge loop • Hyper-activation Self-destruction Highlights d Autoinhibition of HECT ubiquitin ligases by a linker segment d Phosphorylation of linker or cancer associated mutation relieves autoinhibition d Complete loss of linker leads to hyperactivation and self- destruction of ligase Chen et al., 2017, Molecular Cell 66, 345–357 May 4, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2017.03.020 Molecular Cell Article A Tunable Brake for HECT Ubiquitin Ligases Zan Chen,1 Hanjie Jiang,1 Wei Xu,1 Xiaoguang Li,2 Daniel R. Dempsey,1 Xiangbin Zhang,3 Peter Devreotes,2 Cynthia Wolberger,3,5 L. Mario Amzel,3,5 Sandra B. Gabelli,3,4,5,* and Philip A. Cole1,5,6,* 1Department of Pharmacology and Molecular Sciences 2Department of Cell Biology 3Department of Biophysics and Biophysical Chemistry 4Department of Medicine 5Department of Oncology John Hopkins School of Medicine, Baltimore, MD 21205, USA 6Lead Contact *Correspondence: [email protected] (S.B.G.), [email protected] (P.A.C.) http://dx.doi.org/10.1016/j.molcel.2017.03.020 SUMMARY Of the 28 human HECT domain E3 ligases, the most intensively studied comprise the nine members of the NEDD4 family (Fig- The HECT E3 ligases ubiquitinate numerous tran- ure S1A) (Bernassola et al., 2008; Buetow and Huang, 2016; scription factors and signaling molecules, and their Scheffner and Kumar, 2014). Members of the NEDD4 family activity must be tightly controlled to prevent cancer, include WWP2, WWP1, ITCH, and NEDD4-1, and these E3 immune disorders, and other diseases. In this study, ligases target for destruction key signaling molecules and tran- we have found unexpectedly that peptide linkers scription factors (Aki et al., 2015; Buetow and Huang, 2016; tethering WW domains in several HECT family mem- Chen et al., 2014; Scheffner and Kumar, 2014; Zhi and Chen, 2012). Abnormal activities of NEDD4 E3 ligases are connected bers are key regulatory elements of their catalytic ac- to cancer, immune disorders, and other diseases (Aki et al., tivities. Biochemical, structural, and cellular analyses 2015; Broix et al., 2016; Buetow and Huang, 2016; Chen et al., have revealed that the linkers can lock the HECT 2014; Scheffner and Kumar, 2014; Zhi and Chen, 2012). The domain in an inactive conformation and block the NEDD4 family proteins each contain an N-terminal C2 domain proposed allosteric ubiquitin binding site. Such followed by two to four WW domains and culminate in a C-termi- linker-mediated autoinhibition of the HECT domain nal catalytic HECT domain (Figures 1A and S1A) (Buetow and can be relieved by linker post-translational modifica- Huang, 2016). The C2 and WW domains have been implicated tions, but complete removal of the brake can induce in substrate selectivity and catalytic regulation of NEDD4 E3 hyperactive autoubiquitination and E3 self destruc- ligases (Bruce et al., 2008; Escobedo et al., 2014; Mari et al., tion. These results clarify the mechanisms of several 2014; Riling et al., 2015; Wiesner et al., 2007). HECT protein cancer associated mutations and pro- Prior structural studies have revealed that HECT domains contain a larger N-lobe which can interact with E2 proteins and vide a new framework for understanding how HECT a smaller C-lobe that contains a catalytic Cys residue involved ubiquitin ligases must be finely tuned to ensure in ubiquitin transfer (Buetow and Huang, 2016). In addition, normal cellular behavior. several HECT family members have been shown to possess an N-lobe ubiquitin binding exosite (Kim et al., 2011; Maspero et al., 2011; Zhang et al., 2016). Remote from the ubiquitin sub- INTRODUCTION strate binding site in the HECT C-lobe, this exosite has been characterized structurally and shown to engage a distinct ubiq- HECT domain ubiquitin transferases (E3 ligases) catalyze the Lys uitin molecule in HECTs, and this can catalytically activate these ubiquitination of numerous cellular proteins and are critical for E3 ligases (Kim et al., 2011; Maspero et al., 2011, 2013; Ogunjimi protein homeostasis and cell signaling (Buetow and Huang, et al., 2010; Zhang et al., 2016). The N- and C-lobes are con- 2016; Scheffner and Kumar, 2014). Like all E3 ligases, HECT en- nected by a hinge loop, and the HECT domains have been zymes are activated by the participation of upstream E1 and E2 captured in two conformational states, a ground state T-shape enzymes. In contrast to the RING family of E3 ligases, which have (Maspero et al., 2011; Verdecia et al., 2003) and a catalytically an indirect role in ubiquitin bond formation, HECT domains have proficient L-shape (Kamadurai et al., 2013; Maspero et al., an active site Cys that is charged with ubiquitin by the E2. The 2011). The dynamic interconversion of the T-shape and L-shape HECT thioester intermediate directly ubiquitinates target pro- HECT conformations is necessary for turnover, but what governs teins and itself on Lys residues. Because of their direct role in this transition is uncertain. Interactions with the C2 domain, WW catalysis, it is presumed that HECT E3 ligases must be held in domains, and various post-translational modifications have check to prevent both excessive target ubiquitination, as well each been proposed to influence HECT domain catalytic activity, as self-destruction by autoubiquitination (Broix et al., 2016; Bue- but biochemical details for this have generally been lacking tow and Huang, 2016). (Bruce et al., 2008; Gallagher et al., 2006; Gao et al., 2004; Molecular Cell 66, 345–357, May 4, 2017 ª 2017 Elsevier Inc. 345 Figure 1. Full-Length WWP2 Is Autoinhibited (A) Schematic of WWP2 domains with amino acid residue numbers. (B) Ubiquitination assay of GST-tagged WWP2 and GST-free WWP2. The reaction was conducted at 30 C in the presence of 5 mM MgCl2, 5 mM ATP, 100 mM wild-type ubiquitin, 50 nM E1, 1 mM E2 (UbcH5c), and 1 mM E3. The reaction was quenched at 0, 0.5, and 2 hr and samples analyzed by SDS-PAGE followed by colloidal blue staining. The activity of WWP2 was determined by the time-dependent depletion of the unmodified E3 ligase band and the appearance of higher MW bands presumed to represent poly-ubiquitination. (C) Ubiquitination assay of GST-WWP2, full-length WWP2 (FL-WWP2), and WWP2 DC2. The reaction was conducted in the same condition as Figure 1B and quenched at the times indicated. (D) Schematic diagram showing that a TEV protease cleavage site (ENLYFQ/G) was inserted by mutagenesis between residues Ser395 and Ala396 just N-terminal to the WW3 domain. This purified TEV-site containing WWP2 protein was cleaved by TEV protease into a N-terminal fragment X containing the C2 domain, the WW1 domain, and the WW2 domain and a C-terminal fragment Y containing the WW3 domain, the WW4 domain, and the HECT domain. (E) Size-exclusion chromatogram of the TEV-cleaved WWP2 (solid line trace) superimposed with MW standards (dashed line trace). There were two major peaks, Peak A (100 kDa) and Peak B (50 kDa). (legend continued on next page) 346 Molecular Cell 66, 345–357, May 4, 2017 Persaud et al., 2014; Riling et al., 2015; Scheffner and Kumar, importance of WW domain engagement in WWP2 catalytic 2014; Wiesner et al., 2007; Yang et al., 2006). regulation. Here, we investigate the regulation of WWP2, a NEDD4 family We next introduced a protease site N-terminal to the WW3 member that has been shown to ubiquitinate and target for domain in WWP2 (Figure 1D) and determined that the WW3- removal the key tumor suppressor PTEN and the important WW4-HECT protein fragment could partially co-elute with the stem cell-related transcription factor OCT4 (Maddika et al., C2-WW1-WW2 fragment by size exclusion chromatography 2011; Xu et al., 2009). WWP2 contains four WW domains (Figures 1E and 1F). This raised the possibility that interactions (WW1–WW4) and is most closely related to the NEDD4 family between the N-terminal and C-terminal components of WWP2 members WWP1 and ITCH, which have been linked to cancer might modulate the activity of intact full-length WWP2. Consis- and immunologic control (Bernassola et al., 2008; Chang et al., tent with this hypothesis, purified recombinant WWP2 HECT 2006; Zhi and Chen, 2012). Our studies have revealed an unan- domain and WW3-WW4-HECT protein showed robust autoubi- ticipated autoinhibitory module centrally located in WWP2, quitination activity (Figure 1G). Based on these findings, we sus- WWP1, and ITCH between the WW2 and WW3 domains, and pected that the WW1-WW2 moiety was crucial for enforcing below we describe the mechanism of this regulation and its autoinhibition of WWP2. However, intermolecular addition of a general significance in the NEDD4 family. purified recombinant WW1-WW2 protein fragment to WW3- WW4-HECT WWP2 did not inhibit the latter’s ubiquitin trans- RESULTS ferase activity (Figure S2C). WWP2 E3 Ligase Activity Is Autoinhibited The WW2-WW3 Linker, 2,3-Linker, as an Autoinhibitory In the course of in vitro analysis of full-length WWP2 and ubiq- Module of WWP2 uitination of its protein substrate PTEN (Chen et al., 2016), we Because of the lack of an apparent role for the C2 or WW observed that the fusion protein glutathione S-transferase- domains in WWP2 catalytic regulation, we considered the possi- WWP2 (GST-WWP2) was a robust catalyst (Figures 1Band bility that the 30 amino acid (aa) WW2-WW3 linker (2,3-linker), S1B).