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Oct 3, 2017 - The linear ubiquitin chain assembly complex (LUBAC) is the sole identified E3 ligase complex that catalyzes the formation of linear ubiquitin ...
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Structural Insights into SHARPIN-Mediated Activation of HOIP for the Linear Ubiquitin Chain Assembly Graphical Abstract

Authors Jianping Liu, Yingli Wang, Yukang Gong, Tao Fu, Shichen Hu, Zixuan Zhou, Lifeng Pan

Correspondence [email protected]

In Brief LUBAC mediates the formation of linear ubiquitin chains and plays critical roles in numerous signaling pathways. Liu et al. determine the crystal structure of HOIP in complex with SHARPIN and examine the molecular mechanism governing the interaction between two LUBAC components.

Highlights d

The UBA domain of HOIP is responsible for its interaction with SHARPIN

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The molecular mechanism of the interaction between SHARPIN and HOIP is revealed

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SHARPIN and HOIL-1L synergistically bind to different sites of the HOIP UBA domain

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The binding of SHARPIN or HOIL-1L facilitates the E2 loading of HOIP

Liu et al., 2017, Cell Reports 21, 27–36 October 3, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.09.031

Data and Software Availability 5X0W

Cell Reports

Report Structural Insights into SHARPIN-Mediated Activation of HOIP for the Linear Ubiquitin Chain Assembly Jianping Liu,1 Yingli Wang,1 Yukang Gong,1 Tao Fu,1 Shichen Hu,1 Zixuan Zhou,1 and Lifeng Pan1,2,* 1State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, People’s Republic of China 2Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2017.09.031

SUMMARY

The linear ubiquitin chain assembly complex (LUBAC) is the sole identified E3 ligase complex that catalyzes the formation of linear ubiquitin chain, and it is composed of HOIP, HOIL-1L, and SHARPIN. The E3 activity of HOIP can be effectively activated by HOIL-1L or SHARPIN, deficiency of which leads to severe immune system disorders. However, the underlying mechanism governing the HOIP-SHARPIN interaction and the SHARPIN-mediated activation of HOIP remains elusive. Here, we biochemically and structurally demonstrate that the UBL domain of SHARPIN specifically binds to the UBA domain of HOIP and thereby associates with and activates HOIP. We further uncover that SHARPIN and HOIL-1L can separately or synergistically bind to distinct sites of HOIP UBA with induced allosteric effects and thereby facilitate the E2 loading of HOIP for its activation. Thus, our findings provide mechanistic insights into the assembly and activation of LUBAC. INTRODUCTION Ubiquitination, the covalent conjugation of target proteins with one or multiple 76 amino acid small protein ubiquitin (Ub), is one type of the most important and versatile reversible posttranslational modifications in mammals (Pickart and Eddins, 2004; Weissman, 2001) and plays critical roles in numerous biological processes, including protein degradation, DNA repair, inflammatory response, and cell-cycle progression (Hicke et al., 2005; Kerscher et al., 2006; Pickart and Eddins, 2004). The ubiquitination process in general is a three-step catalytic cascade involving the sequential actions of three enzymes: a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3) (Hershko and Ciechanover, 1998; Kerscher et al., 2006). Ub itself can be ubiquitinated to form eight distinct types of homotypic poly-Ub chains, which are interlinked through any one of the seven Lys residues (K6, K11, K27, K29, K33, K48, and K63) or the extreme N-terminal Met1 residue of Ub (Hicke et al., 2005; Iha et al., 2008). As a unique type of poly-Ub chain, the linear poly-Ub chain (also called

the M1-linked poly-Ub chain), in which the backbone carboxyl group of the extreme C-terminal Gly76 of one Ub is conjugated to the backbone amino group of the Met1 residue in the preceding Ub, is widely involved in both innate and adaptive immune signaling pathways (Ikeda, 2015; Iwai et al., 2014; Kirisako et al., 2006; Rieser et al., 2013; Shimizu et al., 2015). Recently, accumulating evidence emphasizes crucial roles of linear polyubiquitination in the activation of various immune and inflammatory receptors and their downstream signaling pathways (Fiil and Gyrd-Hansen, 2014; Ikeda, 2015; Shimizu et al., 2015; Tokunaga, 2013). A panel of important signaling proteins, such as NEMO and RIP1, have been identified to be modified by linear Ub chains (Inn et al., 2011; Iwai et al., 2014; O’Donnell et al., 2007; Peltzer et al., 2016; Rieser et al., 2013; Shimizu et al., 2015; Tokunaga et al., 2009). The linear Ub chain assembly complex (LUBAC), which consists of three proteins, HOIP, HOIL-1L, and SHARPIN, is the only currently known E3 complex responsible for the linear Ub chain formation (Gerlach et al., 2011; Ikeda et al., 2011; Kirisako et al., 2006; Tokunaga et al., 2011). Importantly, malfunction of any of the three LUBAC subunits causes inflammation, immunodeficiency, or even death in animal models and/or humans (Boisson et al., 2012; Gijbels et al., 1996; Peltzer et al., 2014; Sasaki et al., 2013; Tokunaga et al., 2009). As the catalytic subunit of LUBAC, HOIP is a RING-betweenRING (RBR) type E3 (Kirisako et al., 2006; Smit et al., 2012), containing mainly an N-terminal PNGase/UBA or UBX-containing protein (PUB) domain followed by a B-box type zinc finger (ZF), a canonical ZF, two Nlp4-like ZF domains (NZF1 and NZF2), a middle atypical Ub-associated domain (UBA), and a C-terminal RBR domain conjugated with a unique linear Ub chain determining domain (LDD) (Figure 1A). The RBR domain together with the LDD region (RBR-LDD) is the catalytic core of HOIP and is responsible for the conjugation of linear Ub chains (Smit et al., 2012; Stieglitz et al., 2013). Interestingly, the isolated HOIP is in a partially auto-inhibited state, and the UBA domain of HOIP, which cannot recognize Ub proteins, somehow inhibits the catalytic activity of HOIP RBR-LDD (Kirisako et al., 2006; Smit et al., 2012; Stieglitz et al., 2012b). Strikingly, the auto-inhibition of HOIP can be relieved by HOIL-1L and SHARPIN (Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2009, 2011), two accessory subunits of LUBAC. Currently, it is well established that the binding of either HOIL-1L or SHARPIN to HOIP would release the auto-inhibition of HOIP, but so far how the

Cell Reports 21, 27–36, October 3, 2017 ª 2017 The Author(s). 27 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. The UBL Domain of SHARPIN Specifically Binds to the UBA Domain of HOIP (A) A schematic diagram showing the domain architectures of SHARPIN, HOIP, and HOIL-1L proteins as well as the interactions between the three subunits of LUBAC. (B) Mapping the binding region of HOIP for the interaction with SHARPIN UBL domain via co-immunoprecipitation assay. (C) Analytical gel filtration chromatography analyses show the direct interaction between SHARPIN UBL domain and HOIP UBA domain. (D) ITC measurements of the binding affinities of HOIP UBA domain with SHARPIN UBL domain and the full-length SHARPIN protein. (E) Ribbon representation showing the overall structure of SHARPINUBL/HOIPUBA complex. In this drawing, the SHARPINUBL domain is shown in blue and HOIPUBA in green. (F) The combined ribbon and surface representation showing the overall architecture of SHARPINUBL/HOIPUBA complex with the same color scheme as in (A). See also Figure S1 and Table S1.

UBA domain of HOIP poses the inhibition to the RBR-LDD region remains elusive. Therefore, the molecular mechanism underlying the SHARPIN- and HOIL-1L-mediated activation of HOIP remains to be elucidated.

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The HOIL-1L subunit contains a C-terminal RBR domain (Figure 1A), but the catalytic activity of the HOIL-1L RBR domain is not involved in the linear Ub chain assembly (Kirisako et al., 2006). In addition, HOIL-1L has an N-terminal Ub-like (UBL)

domain (Figure 1A) and a middle NZF domain that can specifically recognize linear Ub chains (Sato et al., 2011). Similar to HOIL-1L, SHARPIN contains a UBL domain and a Ub-binding NZF domain (Ikeda et al., 2011) (Figure 1A), but unlike HOIL-1L, SHARPIN uniquely has a N-terminal pleckstrin homology (PH) domain (Stieglitz et al., 2012a). Previously, the interaction between HOIL-1L and HOIP was well characterized, and specifically, the UBA domain of HOIP directly binds to the UBL domain HOIL-1L to mediate the HOIP/HOIL-1L complex formation (Yagi et al., 2012). In contrast, the molecular mechanism governing the interaction of HOIP with SHARPIN is still elusive. Particularly, whether the UBA or the NZF2 domain of HOIP is responsible for the interaction with SHARPIN remains a subject of debate in the literature (Gerlach et al., 2011; Ikeda et al., 2011; Tokunaga et al., 2011). Given the important roles played by SHARPIN in the LUBAC, how SHARPIN interacts with HOIP is a very fundamental question that needs to be addressed. In this study, we present the crystal structure of SHARPIN UBL in complex with HOIP UBA domain and uncover the molecular mechanism underpinning the specific interaction between SHARPIN and HOIP. We further demonstrate that SHARPIN and HOIL-1L can separately or synergistically bind to different sites of HOIP UBA domain with induced allosteric effects, thereby likely causing a conformational re-arrangement between UBA and the RBR-LDD region to facilitate the E2 loading of HOIP for the subsequent linear Ub chain assembly. RESULTS The SHARPIN UBL Domain Specifically Binds to the UBA Domain of HOIP To clarify the interaction mechanism between HOIP and SHARPIN, we first used the GFP-tagged SHARPIN UBL domain (residues 206–309) (hereafter referred to as SHARPINUBL) to map its binding region in HOIP by co-immunoprecipitation (coIP) assay. The results showed that in co-transfected mammalian cells, the UBL domain of SHARPIN could be readily co-precipitated by the full-length HOIP and the HOIP C-terminal fragment (residues 480–1,072) as well as the HOIP UBA domain (residues 480–639) (hereafter referred to as HOIPUBA), but not the HOIP N-terminal fragment (residues 1–479) containing the NZF2 domain or the C-terminal RBR-LDD region (residues 640–1,072) (Figure 1B), indicating that SHARPINUBL only specifically binds to the UBA domain rather than the NZF2 domain of HOIP. Next, using purified recombinant proteins and analytical gel filtration chromatography analysis, we confirmed the direct interaction between SHARPINUBL and HOIPUBA (Figure 1C). Finally, we also quantitatively characterized the interactions of HOIPUBA with SHARPINUBL and the full-length SHARPIN (hereafter referred to as SHARPINFL) by measuring their binding affinities using isothermal titration calorimetry (ITC) and found that SHARPINUBL and SHARPINFL bind to HOIPUBA with similar dissociation constant (KD) values, 3.7 and 7.5 mM, respectively (Figure 1D), revealing that SHARPINUBL alone is sufficient for binding to HOIPUBA. Overall Structure of the SHARPINUBL/HOIPUBA Complex To further uncover the molecular basis of SHARPIN and HOIP interaction, we sought to determine the crystal structure of

the SHARPINUBL/HOIPUBA complex. After extensively crystallization condition screening for the SHARPINUBL/HOIPUBA complex, we successfully obtained good crystals that diffracted to 3.0 A˚ on the synchrotron beamline. The structure of SHARPINUBL/HOIPUBA complex was determined using the single-wavelength anomalous diffraction method with a Se-Met derivative dataset (Table S1). In each asymmetric unit, there are four SHARPINUBL/HOIPUBA complexes, each of which has a 1:1 binding stoichiometry and forms an overall elongated architecture (Figure 1E). In the complex structure, the HOIPUBA domain adopts an extended seven-helix-bundle conformation (Figures 1E and S1A), which resembles a string of three tandem canonical UBAlike domains (Figure S2A), and the SHARPINUBL domain forms a typical Ub/b-grasp fold, containing a five-stranded b sheet, a 3.5-turn a-helix, and a short 310 helix (Figures 1E and S1B). Only the N-terminal a1 and a2 helices of HOIPUBA are directly involved in the SHARPIN binding, and packs against a solvent-exposed patch located in the central b sheet of SHARPINUBL that resembles the canonical I44-containing site in Ub, and buries a total surface area of 867 A˚2 (Figure 1F). Notably, although another contacting site, which consists primarily of the extreme C-terminal loop and the a7 helix of HOIPUBA together with a hydrophobic surface formed mainly by the a1, b1, b2, and b5 of SHARPINUBL, was found between the SHARPINUBL and HOIPUBA domains in the asymmetric unit (Figure S2B), and only half of the HOIPUBA molecules in the asymmetric unit show well-defined electron densities of the residues involved in this contacting surface. Further ITC and GST-fusion protein affinity pull-down assay demonstrated that this contacting site is merely induced by crystal packing, as the HOIP deletion mutant that lacks the C-terminal half of the a7 helix (HOIPUBA DCT) still retains its ability to interact with SHARPIN (Figures 2B, 2D, and S3C). The Molecular Interface of the HOIPUBA/SHARPINUBL Complex Detailed structural analysis showed that binding between HOIPUBA and SHARPINUBL is mediated mainly by hydrophobic interactions (Figures 2A and S3A). Particularly, the hydrophobic side chains of L491, V492, I495, P505, and F509 from HOIPUBA pack against a hydrophobic patch formed by the V271, L276, F296, Y298, and L300 residues of SHARPINUBL, and the side chain of HOIP M484 partially occupies a hydrophobic pocket assembled by the side chains of I272 and P294 from SHARPINUBL (Figures 2A and S3A). Moreover, the SHARPINUBL/HOIPUBA complex was further stabilized by additional polar interactions (Figures 2A and S3B). Specifically, the side chain amine group of Q481 located at the N-terminal part of the a1 helix of HOIPUBA forms two hydrogen bonds with the side chain of D293 as well as the backbone carbonyl of D291 from SHARPIN, and the side chain of HOIP E499 located at the C-terminal part of a1 helix forms two hydrogen bonds with the side chain hydroxyl group of SHARPIN Y298 and the backbone amine group of SHARPIN S301 residue (Figure 2A). Two Arg-Glu pairs of salt bridges (Arg496HOIP-Glu226SHARPIN and Arg269SHARPIN-Glu506HOIP) together with a hydrogen bond between the side chain of HOIP R496 and the backbone carbonyl group of SHARPIN D227 further strengthen the HOIPUBA and SHARPINUBL interaction (Figure 2A).

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Figure 2. Detailed Molecular Interface of SHARPINUBL/HOIPUBA Complex and Its Validation by Site-Directed Point Mutations (A) Enlarged stereo view of ribbon-stick model showing the molecular details of the binding interface between SHARPINUBL and HOIPUBA. The salt bridges and hydrogen bonds involved in the binding are indicated as dashed lines. (B) Overlaid ITC data and fitting curves for the titrations between variants of SHARPIN and HOIP proteins. (C) Overlaid ITC data and fitting curves for the titrations between different HOIPUBA mutants and wild-type SHARPINUBL. (D) The measured binding affinities between different HOIP and SHARPIN proteins by ITC assays. N.D., KD value not detectable. (E) GST pull-down assays verify the interaction between full-length SHARPIN and HOIP as well as the key binding interface residue, SHARPIN V271. (F) In vitro linear ubiquitin chain assembly assays showing that the ability of HOIP to assemble linear ubiquitin chains is stimulated by wild-type SHARPIN but not the SHARPIN V271E mutant, which is unable to interact with HOIP. (G) NF-kB luciferase reporter assays using different HOIP, SHARPIN, and HOIL-1L variants. All luciferase activities are normalized to that of the control cells. Error bars denote the standard deviation between three replicates. See also Figures S2 and S3.

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Figure 3. HOIPUBA Domain Adopts Different Conformations to Interact with SHARPIN and HOIL-1L (A) Overlaid structures of SHARPINUBL/HOIPUBA and HOIL-1LUBL/HOIPUBA complexes in the ribbon representation show the different interaction modes of HOIPUBA in binding to SHARPINUBL and HOIL-1LUBL. In this drawing, the HOIL-1LUBL and HOIPUBA in the HOIL-1LUBL/HOIPUBA complex are drawn in orange and in magenta, respectively. (B) Structural comparison of the configurations of HOIPUBA in the SHARPIN-bound and HOIL-1Lbound states shows the overall re-arrangements of HOIPUBA in binding to SHARPINUBL and HOIL-1LUBL. (C) Comparison of the topologies of HOIPUBA domain in the SHARPIN- and HOIL-1L-bound states. SHARPIN-binding induces the re-arrangement of three a helices (a1, a2, and a3) in the N terminus of HOIPUBA, which are collectively referred as Module1. Asterisks indicate the positions of the three critical hydrophobic residues (M484, V492, and I495) involved in SHARPIN binding. See also Figure S4.

Consistent with our structural data, ITC results showed that the substitution of the conserved V492 or I495 residue of HOIP or the V271 residue of SHARPIN with a Glu residue all completely abolished the SHARPINUBL/HOIPUBA complex formation (Figures 2B, 2D, and S1), and the replacement of Q481, R496, E499, or E506 residue of HOIP with an Ala residue reduced the interaction between SHARPINUBL and HOIPUBA (Figures 2C and 2D). Using the GST-fusion protein affinity pull-down assay, we further verified the specific interactions between HOIP and SHARPIN observed in the complex structure (Figures S3C and 2E). Mutations of key interface residues of the SHARPINUBL/HOIPUBA complex, such as the V492E and I495E mutations of HOIP and the V271E mutation of SHARPIN, all essentially abolished the specific interaction between HOIPUBA and SHARPIN (Figure S3C). In contrast, the L624E mutation of HOIP UBA domain, which could disrupt the binding between HOIP UBA and HOIL-1L (Yagi et al., 2012), retained its ability to interact with SHARPIN (Figure S3C). Importantly, the V271E mutation of SHARPIN also completely disrupted the binding between full-length SHARPIN and HOIP (Figure 2E), further confirming our conclusion that the specific interaction between HOIPUBA and SHARPINUBL is essential for the HOIP/SHARPIN complex formation. The Specific Interaction between SHARPINUBL and HOIPUBA Is Essential for the SHARPIN-Mediated Activation of HOIP Previous studies showed that there is a reciprocal inhibition between HOIP UBA domain and the RBR-LDD region (Kirisako et al., 2006; Smit et al., 2012). Using in vitro linear Ub chain assembly assays, we confirmed this notion and further demonstrated that either SHARPINUBL or SHARPINFL can efficiently activate the activity of HOIP to assemble linear Ub chain (Figures S3D and 2F), while in contrast, the V271E mutant of SHARPINUBL or SHARPINFL, which lost its ability to interact with HOIPUBA (Figure 2B), was unable to promote the activity of HOIP (Figures S3D and 2F), revealing that the specific interaction between SHARPIN and HOIPUBA is essential for the SHARPIN-mediated

activation of HOIP’s enzyme activity. Because the linear ubiquitination is crucial for activation of the NF-kB pathway, to further investigate the specific function of SHARPINUBL and HOIPUBA interaction for the activation of LUBAC in cells, we also established an NF-kB reporter-based luciferase assay. The results showed that co-expression of HOIP together with SHARPIN leads to a strong activation of the NF-kB pathway, while expression of HOIP or SHARPIN alone has weak or negligible effects (Figure 2G). In agreement with our in vitro biochemical and structural analyses, disruption of the specific interaction between HOIP and SHARPIN by single-residue mutation either on HOIP (HOIP V492E mutation) or SHARPIN (SHARPIN V271E mutation) largely attenuated or essentially abolished the activation of NF-kB pathway in co-transfected cells (Figure 2G). As controls, the HOIP V492E and SHARPIN V271E mutations, which do not affect the interaction between HOIL-1L and HOIP, had no obvious effects on the HOIL-1L and HOIP-mediated activation of NF-kB pathway (Figure 2G). HOIPUBA Adopts Different Conformers to Interact with SHARPINUBL and HOIL-1LUBL Previously, the HOIP UBA domain was demonstrated to interact with the HOIL-1L UBL domain, and the determined HOIL-1LUBL/ HOIPUBA complex structure showed that HOIL-1LUBL specifically binds to the last C-terminal a-helix of HOIPUBA domain (Yagi et al., 2012) (Figure S4A). Interestingly, detailed sequence alignment and structural comparison analyses showed that SHARPINUBL and HOIL-1LUBL share a high similarity, and except for a longer loop between the b1 and b2 strands, SHARPINUBL has highly similar or identical residues corresponding to the ones in the HOIL-1LUBL that are responsible for HOIPUBA binding (Figures S4B and S4C). However, the interaction mode of SHARPINUBL/HOIPUBA complex is totally distinct from that of the HOIL-1LUBL/HOIPUBA complex (Figure 3A). Notably, in the SHARPINUBL/HOIPUBA complex, SHARPINUBL uses its solventexposed surface of the central b sheet to interact with the N-terminal a1 helix of HOIPUBA, while in the HOIL-1LUBL/HOIPUBA Cell Reports 21, 27–36, October 3, 2017 31

(legend on next page)

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complex, the extreme C-terminal part of HOIPUBA directly contacts a unique pocket of HOIL-1LUBL that is assembled mainly by the a1, b1, b2, and b5 of HOIL-1LUBL (Figures 3A and S4D). In addition, the overall structural topologies of the HOIP UBA domain in the SHARPINUBL/HOIPUBA and HOIL-1LUBL/HOIPUBA complexes are remarkably different (Figures 3B and 3C). Particularly, the N-terminal three a helices (a1, a2, and a3) of HOIPUBA in the HOIL-1LUBL/HOIPUBA complex are re-arranged into an extended conformation in the SHARPINUBL/HOIPUBA complex, and the extreme C-terminal a9 helix of HOIPUBA in the HOIL1LUBL/HOIPUBA complex is mainly un-structured in the SHARPINUBL/HOIPUBA complex (Figures 3B and 3C). As the a1, a2, and a3 helices of HOIPUBA in the HOIL-1LUBL/HOIPUBA complex are arranged into a canonical UBA-like fold (Figure S4A), and this region (hereafter referred to as Module1, residues 480–526) contains the SHARPINUBL-binding sequences (Figure S1A), we wondered whether this module alone may bind to SHARPINUBL. Therefore, we isolated this module and tested its binding to SHARPINUBL. Interestingly, GST-fusion protein affinity pull-down assay and ITC-based analysis revealed that it cannot bind to SHARPINUBL (Figures S3C and 2D). The inability of isolated Module1 to interact with SHARPINUBL strongly indicated that the Module1 is structurally coupled with the middle part of HOIPUBA domain, and the intact UBA domain of HOIP is essential for the SHARPINUBL binding. SHARPINUBL and HOIL-1LUBL May Synergistically Bind to HOIP with Induced Allosteric Effects Because our structural data showed that SHARPINUBL and HOIL-1LUBL can differentially bind to distinct regions of the HOIPUBA domain, we next sought to determine the relationship between SHARPINUBL and HOIL-1LUBL in binding to HOIPUBA. We had set up two biochemical assays on the basis of analytical gel filtration chromatography followed by SDS-PAGE analyses using the purified HOIPUBA/HOIL-1LUBL complex and the HOIPUBA/SHARPINUBL complex mixed with increasing amounts of SHARPINUBL and HOIL-1LUBL protein, respectively (Figures 4A, 4B, S5A, and S5B). The analytical gel filtration chromatography results showed that with increasing amounts of SHARPINUBL or HOIL-1LUBL proteins added into the HOIPUBA/ HOIL-1LUBL or the HOIPUBA/SHARPINUBL complex, the absorbance at 280 nm (A280) of the complex peak becomes much higher, and the complex peak elutes earlier (Figures 4A and S5A), indicating that a larger protein complex forms in the pres-

ences of HOIPUBA, SHARPINUBL, and HOIL-1LUBL proteins. Indeed, the corresponding SDS-PAGE results (fraction 1) together with analytical ultracentrifugation analyses confirmed that SHARPINUBL and HOIL-1LUBL can simultaneously interact with HOIPUBA to form a ternary complex (Figures 4B, S5B, and S5C). Interestingly, further ITC-based analyses showed that SHARPINUBL binds to a longer HOIP fragment (residues 480– 1,072), which encompasses the UBA, RBR, and LDD domains, with a much weaker binding affinity than that to the isolated HOIPUBA (Figures 4C and 1D). Intriguingly, the longer HOIP fragment showed a restored binding affinity toward SHARPINUBL (with a KD of 5.3 ± 0.1 mM), once it was complexed with HOIL1LUBL (Figure 4D). Conversely, a similar phenomenon was also observed when comparing the ITC results of HOIL-1LUBL titrated with the purified HOIPUBA, HOIP(480–1,072) and the HOIP(480– 1,072)/SHARPINUBL complex, respectively (Figures S5D–S5F). These data clearly indicate that there is a reciprocal inhibition between the UBA domain and the RBR-LDD region of HOIP, and SHARPIN and HOIL-1L can both release this auto-inhibition and synergistically interact with HOIP. Importantly, although the UBL domain of SHARPIN or HOIL-1L is sufficient to stimulate the activity of HOIP to assemble linear Ub chain either using the E2 enzyme UBE2L3 or UBE2D1 (Figures 4E and S6A), the synergistic binding of these two UBL domains to HOIPUBA activates HOIP more potently than either one individually (Figures 4E, 4F, and S6). Consistently, the co-expression of HOIP together with SHARPIN and HOIL-1L leads to a much stronger activation of the NF-kB pathway than that of the co-expression of HOIP with either SHARPIN or HOIL-1L in the luciferase assay (Figure 2G). The synergistic effect implies that HOIP UBA domain undergoes an allosteric conformational change during SHARPINUBL or HOIL-1LUBL binding, which can be readily monitored by protein nuclear magnetic resonance (NMR) experiments. In the 1H-13C HSQC spectrum, the NMR peaks of ε1-methyl group (ε1-CH3) from methionine residues and d1-methyl group (d1-CH3) of isoleucine residues are located in two characteristic regions that are well separated from other CH groups. Coincidentally, all the methionine and isoleucine residues in HOIPUBA are far away from the HOIL-1LUBL binding region but within or close to the SHARPINUBL-binding site (Figures S1A and S7A). Therefore, it was feasible for us to follow the potential allosteric effects on the SHARPIN-binding region of HOIPUBA induced by

Figure 4. SHARPINUBL and HOIL-1LUBL Can Synergistically Bind to and Cooperatively Activate HOIP by Facilitating Its E2 Loading (A) Analytic gel filtration chromatography analyses of the HOIPUBA/HOIL-1LUBL complex incubated with increasing molar ratio of SHARPINUBL proteins. (B) SDS-PAGE combined with Coomassie blue staining analyses shows the protein components of corresponding fraction 1 and fraction 2 collected from different analytic gel filtration chromatography experiments in (A). (C and D) ITC measurement of the binding affinity of SHARPINUBL to HOIP(480–1,072) (C) and HOIL-1LUBL/HOIP(480–1,072) complex (D). (E) In vitro linear ubiquitin chain assembly assays using purified E1, E2 enzyme UBE2L3, Ub, HOIP(299–1,072), HOIL-1LUBL, and SHARPINUBL proteins show that HOIL-1LUBL and SHARPINUBL can cooperatively activate HOIP to assemble linear ubiquitin chains. Asterisks indicate the bands of degraded E1 and HOIP proteins. (F) Quantitative measurements of the linear ubiquitin chain synthesis activities by quantification of the amount of residual mono-Ub at each time point under different protein complex conditions in (E). The concentration of mono-Ub is represented by the gray-level integration of mono-Ub band in each lane on the gel and normalized by the value at 0 min for each reaction condition. (G–I) ITC measurement of the binding affinity of UBE2L3 to HOIP(480–1,072) (G), the HOIP(480–1,072)/SHARPINUBL complex (H), and the HOIP(480–1,072)/HOIL1LUBL complex (I). See also Figures S5–S7.

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HOIL-1L binding. The NMR signals of these characteristic methyl groups from the methionine and isoleucine residues of HOIPUBA showed multiple peaks, mainly because of the dynamic nature of the isolated HOIPUBA (Figure S7B). Interestingly, all these types of NMR peaks were perturbed in the presence of HOIL1LUBL (Figure S7B), indicating that the whole HOIP UBA domain undergoes substantial conformational changes when binding to HOIL-1LUBL. The Binding of HOIP with SHARPIN or HOIL-1L Facilitates Its E2 Loading Because our data showed that the binding of SHARPINUBL or HOIL-1LUBL domain to HOIP is sufficient to disrupt the reciprocal inhibition between the UBA domain and RBR-LDD region of HOIP, and to stimulate the activity of HOIP to assemble linear Ub chain (Figure 4E), we wondered what happens in the RBRLDD region when the UBA domain of HOIP is engaged with SHARPIN or HOIL-1L. We used the E2 UBE2L3 as a cognate probe to test the accessibility of HOIP RBR-LDD region. Our ITC results showed that the apo form of UBA-RBR-LDD protein has a weak binding affinity for the E2 (KD value 42 mM), and the binding of either SHARPINUBL or HOIL-1LUBL to HOIP UBA-RBRLDD protein increases its binding affinity for E2 about 4–7 times (KD values 10.5 and 5.8 mM, respectively) (Figures 4G–4I). These data indicated that HOIP is in an auto-inhibited state, and the binding of either SHARPIN or HOIL-1L to the HOIP UBA domain can ‘‘open’’ this state to facilitate its E2 binding. DISCUSSION In this study, using ‘‘solid’’ biochemical and structural analyses, we elucidated the molecular mechanism governing the specific interaction between HOIP and SHARPIN and demonstrated that SHARPIN directly binds to the UBA domain rather than the NZF2 domain of HOIP. Because the purified NZF2 domain of HOIP is unable to directly interact with the SHARPIN UBL domain (Figure S7C), the previously reported inconsistence of whether the UBA or the NZF2 domain of HOIP is responsible for the interaction between HOIP and SHARPIN is likely due to an indirect association mediated by the HOIP NZF2. In addition, our study also revealed that HOIP could separately or synergistically bind to SHARPIN and HOIL-1L via two distant binding sites in its UBA domain, thereby providing a mechanistic explanation to the assembly of three LUBAC-related complexes: the HOIP/HOIL-1L and HOIP/SHARPIN dimeric complexes as well as the SHARPIN/HOIP/HOIL-1L trimeric complex. Interestingly, previous studies showed that the HOIP UBA domain does not directly bind to the catalytic RBR-LDD region of HOIP (Stieglitz et al., 2013), but it somehow indirectly couples with the RBR-LDD region and keeps it in an auto-inhibited state (Smit et al., 2012; Stieglitz et al., 2012b; Tokunaga et al., 2009). In this study, we revealed that the binding of SHARPINUBL or HOIL1LUBL to the UBA domain of HOIP could release the auto-inhibition of HOIP (Figure 4E). Although SHARPINUBL and HOIL-1LUBL bind to different regions of HOIP UBA domain (Figure 3A), both of them could allosterically induce conformational changes in HOIPUBA when binding to HOIP, as indicated by our biochemical and structural results (Figures 3, 4C, 4D, S5E, S5F, and S7B).

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Therefore, it is likely that the binding of SHRAPIN or HOIL-1L to HOIP would drive conformational changes in the UBA domain of HOIP, which in turn allosterically re-arrange the relative orientation between UBA and RBR-LDD, thereby facilitating the E2 loading of the RBR-LDD region and enhancing its catalytic activity. In contrast to a recent study (Stieglitz et al., 2012b), which reported that HOIL-1L and SHARPIN are not additive in promoting the activity of HOIP on the basis of a qualitative in vitro ubiquitination assay using the full-length SHARPIN and/or HOIL-1L, our quantitative results demonstrated that SHARPINUBL and HOIL1LUBL actually could synergistically bind to HOIP and cooperatively stimulate its enzymatic activity to assemble linear Ub chain (Figures 4C–4F and S6). This difference might be due to the different constructs used for the in vitro ubiquitination assay, as the full-length SHARPIN or HOIL-1L might exist in a conformation that could alter its binding affinity to HOIP. We sought to use X-ray crystallography to solve the structure of HOIP UBA-RBR-LDD region, but unfortunately, after numerous trials, we failed to obtain high-quality crystals for structural determinations. Therefore, further work is required to clarify how HOIP is precisely activated by SHARPIN and HOIL-1L. EXPERIMENTAL PROCEDURES Protein Expression and Purification The unlabeled, 13C-labeled, or selenomethionine (Se-Met)-labeled recombinant proteins used in this study were expressed in E. coli BL21 (DE3) and purified by Ni2+-NTA agarose or glutathione Sepharose 4B resin followed by size-exclusion chromatography. The N-terminal Trx-His6 tag or GST-tag was cut by 3C protease and removed by size-exclusion or ionic exchange chromatography. See Supplemental Experimental Procedures for details. Analytical Gel Filtration Chromatography Samples were loaded onto a Superose 12 10/300 GL column connected to an AKTA fast protein liquid chromatography (FPLC) system. Proteins were eluted out in a buffer containing 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM DTT. NMR Spectroscopy The 13C-labeled proteins for NMR studies were concentrated to 0.1 mM in a buffer containing 50 mM NaH2PO4/Na2HPO4, 50 mM NaCl, and 1 mM DTT (pH 7.0). The 1H-13C HSQC spectra were collected at 30 C on an Agilent 800 MHz spectrometer. Crystallography Crystals of HOIPUBA/SHARPINUBL complex were grown at 16 C in the hanging drops over a reservoir buffer containing 0.1 M HEPES (pH 7.3), 7% (w/v) PEG8000, and 8% (v/v) ethylene glycerol. Crystals were harvested in the reservoir buffer with 10% (v/v) glycerol added as the cryo-protectant. The X-ray diffraction dataset was collected at the beamline BL17U1 of the Shanghai Synchrotron Radiation Facility. The complex structure was determined, refined, and validated with the PHENIX suite (Adams et al., 2002). See Supplemental Experimental Procedures for details. GST Pull-Down Assay The GST pull-down assay was performed similarly as described previously (Li et al., 2016). See Supplemental Experimental Procedures for details. In Vitro Ubiquitination Assays Linear ubiquitination chain was assembled at 37 C in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 20 mM MgCl2, and 20 mM ATP. Proteins were mixed together at the following final concentrations: 2 mM Ube1 (E1), 5 mM UBE2L3 (E2), 1.5 mM of HOIP(299–1,072), and 100 mM Ub, with or without

5 mM HOIL-1L or 5 mM SHARPIN. Samples were taken at specified time intervals and resolved by SDS-PAGE gels and visualized by Coomassie blue staining. ITC ITC measurements were carried out on a PEAQ-ITC calorimeter at 25 C. The titration data were analyzed with the PEAQ-ITC analysis software supplied by manufacturer and fitted with the one-set-of-binding-sites model. Analytical Ultracentrifugation Sedimentation velocity experiments were performed on a Beckman XL-I analytical ultracentrifuge equipped with an eight-cell rotor at 20 C. The data were analyzed with the program SEDFIT (http://www.analyticalultracentrifugation. com). Cell Culture, Transfection, and Immunoprecipitation The HEK293T cell culture, related transfection, and immunoprecipitation were performed similarly as described previously (Li et al., 2016). See Supplemental Experimental Procedures for details. Luciferase Assay HEK293T cells were co-transfected with the luciferase reporter plasmids pGL3-NF-kB-Luc and pRL-SV40 and the appropriate LUBAC plasmids using Lipofectamine 2000. After transfection for 24 hr, cells were lysed in the passive lysis buffer, and luminescence was measured by a GloMax 20/20 Luminometer using the Dual-Luciferase reporter assay kit. DATA AND SOFTWARE AVAILABILITY The accession number for the structure of HOIP UBA in complex with SHARPIN UBL reported in this paper is PDB: 5X0W. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and one table and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2017.09.031. AUTHOR CONTRIBUTIONS J.L., Y.W., and L.P. designed the research. J.L. and Y.W. performed the research. Y.G., S.H., Z.Z., and T.F. helped in the data analysis. J.L. and L.P. analyzed the data and wrote the manuscript. ACKNOWLEDGMENTS We thank Shanghai Synchrotron Radiation Facility BL17U1 for X-ray beam time, Dr. Zhijie Lin and Dr. Jianchao Li for helping to collect the X-ray diffraction data, Dr. A’ming Cao for providing the luminometer, and Prof. Mingjie Zhang for critically reading the manuscript. This work was supported by grants from the National Natural Science Foundation of China (NSFC) (21621002), the Ministry of Science and Technology of China (2016YFA0501903 and 2013CB836900), the STCSM (15JC1400400), and the Chinese Academy of Sciences (XDB20000000); a Thousand Talents Program young investigator award (to L.P.); and grants from the NSFC (31500597) and the Shanghai Science and Technology Committee (15ZR1449100) (to J.L.). Received: June 19, 2017 Revised: August 15, 2017 Accepted: September 7, 2017 Published: October 3, 2017 REFERENCES Adams, P.D., Grosse-Kunstleve, R.W., Hung, L.W., Ioerger, T.R., McCoy, A.J., Moriarty, N.W., Read, R.J., Sacchettini, J.C., Sauter, N.K., and Terwilliger, T.C.

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