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Structural Analysis of SHARPIN, a Subunit of a Large Multi-protein E3 Ubiquitin Ligase, Reveals a Novel Dimerization Function for the Pleckstrin Homology Superfold*

Background: SHARPIN is a subunit of the E3 ligase complex LUBAC and the gene that is mutated in chronic proliferative dermatitis mice. Results: The N-terminal portion of SHARPIN adopts the PH superfold and mediates homodimerization. Conclusion: The PH superfold can be used as a protein dimerization module. Significance: This study highlights the versatility of the PH superfold and its function as a protein interaction module.

SHARPIN (also known as Sipl1) is a 387-amino acid protein that originally has been identified as a SHANK (SH3 (Src homology 3) and multiple ankyrin repeat domains protein)binding protein enriched in the postsynaptic density of excitatory neurotransmitters (1). More recently, the observation that a spontaneous mutation in the mouse SHARPIN gene causes chronic proliferative dermatitis, a disease marked by skin lesions, multi-organ inflammation, and a deficient immune system suggested that SHARPIN might also be implicated in immune and inflammatory signaling pathways (2). Such a role has now been confirmed by the discovery that SHARPIN acts as a novel subunit of the E3 ubiquitin ligase complex LUBAC, which catalyzes the formation of linear or M1-linked polyubiquitin chains that play an important role in the activation of the transcription factor NF-B (3)(4)(5). LUBAC contains two other subunits, termed HOIL-1L (also known as RBCK1) and HOIP (HOIL-1L interacting protein, also known as RNF31), both of which are members of the RBR (RINGbetween-RING) subfamily of RING family E3 ligases (6), with the RBR domain residing in the C-terminal portion of the molecules (7)(8)(9)(10). The N-terminal half of HOIL-1L consists of an ubiquitin-like domain (UBL) 2 followed by an Npl4 zinc finger (NZF). The UBL recognizes an ubiquitin-associated domain in HOIP to mediate complex formation (7).
Other domains present in HOIP include a PUB (Peptide: N-glycanase/UBA or UBX-containing proteins) domain followed by a zinc finger (ZF) and two NZF domains. Mutational studies have shown that the RBR domain of HOIP is the active side for linear ubiquitin chain synthesis, whereas the RBR of HOIL-1L is dispensable for catalysis, although complex formation between the two proteins is required for enzymatic activity (7,8). SHARPIN does not contain an RBR domain but shares significant similarity with the N-terminal region of HOIL-1L that comprises the UBL and NZF domains (see Fig. 1A) and similar to HOIL-1L binds ubiquitin via the NZF, whereas its UBL domain is responsible for the interaction with HOIP (3)(4)(5). This related domain architecture and sequence similarity likely explains the apparent overlapping function of the two proteins as SHARPIN can substitute for HOIL-1L in promoting linear ubiquitin chain synthesis by HOIP. Furthermore, the observation that endogenous HOIP can form a complex with only HOIL-1L or SHARPIN in the absence of the other and that either of these complexes can synthesize linear ubiquitin chains to promote NF-B activation indicates that some of the functions of the two proteins are redundant (3,5,8). Nevertheless, clear functional differences exist: it has been shown that SHARPIN but not HOIL-1L deficiency in mice results in an increased TNF␣-induced cell death, whereas HOIL-1L has a much more profound effect on IL-1␤-induced NF-B activation (3)(4)(5). These specific functions of SHARPIN and HOIL-1L are most likely mediated by distinct structural features outside the UBL-NZF segment that is shared between the two proteins. SHARPIN contains a 170-amino acid stretch at the N terminus, which is not well characterized and is absent in HOIL-1L. Immune precipitation experiments showed this region to undergo homomultimerization, an observation that is in line with the presence of a short heptad repeat motif-spanning residues 36 -49 that is predicted to form a coiled coil (1). However, apart from these findings, no further information about the functional or structural properties of the N-terminal part of SHARPIN is currently available. To gain insight into the properties of this region, we have solved the x-ray structure of a SHARPIN construct containing residues 1-127 and characterized its propensity for self-association. The structure confirms that SHARPIN has the ability to self-associate into dimers but rather unexpectedly shows that dimerization is not mediated by a coiled coil but that SHARPIN utilizes the pleckstrin homology (PH) superfold as a dimerization module instead. This mode of interaction is unique and represents a novel function for the PH superfold.

EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification-The N-terminal fragment of human SHARPIN comprising residues 1-127 was expressed in pGEX-4T1 (GE Healthcare). The interface mutant V114D and the mutant L22M/L101M used for producing selenomethionine-substituted protein was generated by site-directed mutagenesis according to the QuikChange protocol (Stratagene) and verified by DNA sequencing. The protein was purified on GSH-Sepharose, and the GST was cleaved with thrombin at 4°C overnight. The cleaved protein was further purified by gel filtration using a Superdex S75 column (GE Healthcare) run in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 1 mM DTT. Purified SHARPIN was concentrated to a final concentration of 5 mM and stored at Ϫ80°C.
Crystallography-Crystallization of SHARPIN 1-127 and SHARPIN 1-127 L22M/L101M will be described in detail elsewhere. In brief, crystals were grown from a 10 mg/ml protein solution in sitting drop set-ups using a reservoir solution containing 4 M sodium formate and optimized by seeding. The mother liquor was not buffered, and its pH was measured to be pH 7.7. Crystals were flash-frozen in liquid nitrogen with a solution containing the mother liquor and 10% glycerol. Data were collected at 100 K. Crystals diffract to 2.0 Å and belong to the primitive tetragonal space group P4 3 2 1 2 with four molecules per asymmetric unit. A data set of high redundancy was collected on beamline I0.4 at the Diamond Light Source (Oxford, UK) and processed using Denzo and Scalepack (11). The structure was solved by single anomalous dispersion phasing using the SeMet derivative SHARPIN 1-127 L22M/L101M. Heavy atom search, density modification, and initial model building was performed using Phenix AutoSol (12). After several rounds of iterative manual model building with Coot (13), the structure was refined using the program REFMAC5 (14), and the strerochemistry of the final model was analyzed with Procheck (15).
Isothermal Titration Calorimetry-Dilution experiments were conducted to investigate the dissociation process of the monomer-dimer transition using an ITC200 calorimeter (GE Healthcare). Protein samples of WT and mutant V114D SHARPIN were prepared by dialysis against 50 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP)). Measurements were performed at 25°C, and data were analyzed using the Origin-based software provided by the manufacturer.
Analytical Ultracentrifugation-Sedimentation velocity experiments were performed in a Beckman Optima XL-I analytical ultracentrifuge using conventional aluminum double-sector centerpieces and quartz windows. Solvent density, viscosity, and the protein partial specific volumes were calculated using SEDNTERP. Prior to centrifugation, samples were prepared by dialysis against the buffer blank solution, 50 mM HEPES, pH 7.4, 150 mM NaCl, and 1 mM TCEP. Samples (400 l) and buffer blanks (420 l) were loaded into the cells, and centrifugation was performed at 50,000 rpm and 293 K in an An-60 Ti rotor. Interference images were collected every 180 s during the sedimentation run. The data were analyzed as a distribution of sedimentation coefficients using SEDFIT (16). Sedimentation equilibrium experiments were performed in a Beckman Optima XLA analytical ultracentrifuge using charcoal-filled epon six-channel centerpieces in an An-60 Ti rotor. Prior to centrifugation, samples were prepared by dialysis against the buffer blank solution, 50 mM HEPES, pH 7.4, 150 mM NaCl, and 1 mM TCEP. The solutions were allowed to reach equilibrium at 18,000, 20,000, 26,000, and 30,000 rpm. To determine the dissociation constant (K d ), the sedimentation equilibrium data were fitted globally with the programm SEDPHAT (17) using a monomer-dimer ideal solution model.
Multi-angle Laser Light Scattering (MALS)-Molecular mass and molecular mass distributions were determined using size exclusion chromatography coupled to multi-angle light scattering. Samples of WT and mutant V114D SHARPIN were applied in a volume of 100 l to a Superdex 75 10/300 GL column (GE Healthcare) equilibrated in 50 mM HEPES, pH 7.4, 150 mM NaCl, and 1 mM TCEP, at a flow rate of 0.5 ml/min. The column was mounted on a Jasco HPLC. The scattered light intensity of the column eluate was recorded using a DAWN-HELEOS laser photometer (Wyatt Technology, Santa Barbara, CA). The protein concentration of the eluent was determined using an OPTILAB-rEX differential refractometer (Wyatt Technology). The weight-averaged molecular mass of the eluent was determined using the ASTRA software (version 5.1; Wyatt Technology). Measurements were performed at 25°C.

RESULTS AND DISCUSSION
Self-association of N-terminal Portion of SHARPIN-SHARPIN contains 387 amino acids with UBL and NZF domains covering residues 219 -377 (Fig. 1A). In contrast, a scan of the region N-terminal to the UBL-NZF module against the PROSITE and SMART databases (18,19) did not provide any hits for recognizable domains. Therefore, to gain threedimensional insight into the N-terminal half of SHARPIN, we screened this region for soluble constructs of bacterially expressed fragments suitable for structural studies. One of the constructs that could be purified to homogeneity covers amino acids 1-127. This construct includes the region spanning amino acids 36 -49 that displays a heptad repeat motif and is predicted with high propensity to form a short coiled coil. To investigate whether SHARPIN 1-127 indeed reversibly self-associates in solution, we performed sedimentation velocity experiments. At a concentration of 38 M, the polypeptide pri-marily exists as a single species with a sedimentation coefficient of 1.3 S, which corresponds to a molecular mass of 13,150 Da, the mass of a SHARPIN monomer (Fig. 1B). However, when the SHARPIN concentration was increased by 2-and 4-fold, respectively, another species with a sedimentation coefficient of 1.9 S became apparent (Fig. 1B). This population corresponds to the molecular mass of a dimer, demonstrating that SHARPIN 1-127 exists in monomer-dimer equilibrium. The high concentrations required to form the dimeric species indicated a rather weak intermolecular interaction, and we were able to derive a dissociation constant of 160 M by sedimentation equilibrium measurements (Fig. 1C). In an alternative approach, we determined the dissociation constant for the monomer-dimer equilibrium by isothermal titration calorime- These data are consistent with the observation that SHARPIN multimerizes through its N-terminal region in vivo (1) and are the first molecular description of this self-association process.
N-terminal Region of SHARPIN Adopts PH Superfold-To understand the structural basis of SHARPIN dimerization, we crystallized the fragment containing residues 1-127. Crystals of space group P4 3 2 1 2 were grown in 4 M sodium formate from selenomethionine-derivatized protein, which diffracted to a maximal resolution of 2.0 Å with four molecules in the asymmetric unit. The structure was solved by single anomalous dispersion (Table 1 and supplemental Fig. S1). Remarkably, the structure of the four subunits in the asymmetric unit revealed that SHARPIN utilizes a PH fold for self-association (Fig. 2, A  and B). Residues 20 -120 of SHARPIN cover the canonical PH fold, which is a seven-stranded antiparallel ␤-sheet, strongly bent to form a ␤-barrel-like conformation and capped by a C-terminal ␣-helix. A helical turn that connects strand ␤4 and ␤5 of the collapsed ␤-barrel is the only variation of the conserved PH domain organization. Residues 1-20 adopt different conformations in the four copies in the asymmetric unit. The N terminus of one of the subunits is buried into the center of the symmetry axis of the tetramer and participates in intermolecular interactions with the other PH-like domains (Fig. 2A). The amino-terminal stretch of the diagonally opposite subunit makes contacts to symmetry related molecules. No density can be observed for the remaining other two N termini, indicating that they are disordered. Each of the protomers in the crystallographic tetramer forms two distinctive interfaces. Because our solution studies did not give any indication that SHARPIN assembles into higher order oligomers, it is likely that tetramerization is due to crystal packing. The subunits I and II are aligned head-to-head against the subunits IV and III, respectively, with a rotation of 180 o degrees ( Fig. 2A). This orientation causes the formation of a histidine-stacking interaction, which is sandwiched between two salt bridges and probably based on crystal contacts (supplemental Fig. S2). In comparison, the second interface (I-II and III-IV) (Fig. 2B) is formed predominantly by a perpendicular arrangement of the helical elements of two neighboring PH-like domains and involves the interplay of many more side chains (Fig. 3, A and B). Val-114 is in the center of a hydrophobic interface formed by Leu-115, Leu-21, and Phe-56 of the other dimer half (Fig. 3, A and B). The hydrophobic patch is limited by electrostatic interactions, which  involve the side chains of two arginines positioned at the C-terminal ␣-helix. Arg-111 binds the backbone oxygen of Leu-21 in ␤1 and Arg-117 coordinates the carbonyl group of Gly-118 and the side chain of Glu-122, which are both located at the tip of the helix. To confirm the relevance of the observed interface for self-association, we introduced a negative charge into the hydrophobic patch of the dimerization side. The mutation V114D should impair the hydrophobic network of the dimer interface and abolish dimerization in solution if this was the physiologically relevant dimer interface. In a complementary approach to our AUC analysis, we performed size exclusion chromatography coupled to multi-angle light scattering measurements of the wild-type and mutant PH-like domain. In these experiments, the retention time of wild-type SHARPIN shifts from a monomeric to a dimeric species when the concentration is increased in line with a monomer-dimer equilibrium (Fig. 4A). In contrast, the V114D mutant elutes as a monomer even at the very high concentration of 2.5 mM. Similarly, no monomer-dimer equilibrium can be observed for V114D when probed by ITC (Fig. 4B). These results clearly indicate that complex formation of the PH-like dimer is indeed formed via the helical interface observed in the crystal structure.
Does N-terminal Portion of SHARPIN Act as Protein Interaction Domain?-The PH superfold is a stable fold that has been adopted by a number of protein interaction domains that otherwise share no sequence homology but use the PH superfold as a scaffold upon which different ligand binding surfaces are crafted (20). These include PH domains themselves, which have been identified as phosphatidylinositol phosphate binding modules and function as membrane-targeting domains (21), as well as for example EVH1 (Enabled/VASP homology) domains, which act as protein-protein interaction modules (22,23). Interestingly, only ϳ15% of all PH domains show specific lipid binding properties (21), whereas the majority bind phospholipid ligands only with low affinity and specificity or show no interaction at all. SHARPIN appears to belong to the latter group as it did not show any detectable phospholipid binding in protein lipid overlay assays (PIP strips, data not shown). In concordance, SHARPIN lacks the electrostatic polarization typically observed for those PH domains that bind to phospholipids and associate with negatively charged membrane surfaces (20) (supplemental Fig. S3, A and B), further supporting the notion that its PH-like domain most likely does not function as a lipidbinding module.
A search of the proteins structure database server DaliLite (version 3) (24) with our structure revealed the EVH1 (Enabled/ VASP homology 1) domain of the scaffold protein HOMER as a close three-dimensional neighbor of SHARPIN (root mean square deviation, 2.6 Å 2 ) (25) (supplemental Fig. S4A). EVH1 domains adopt the same core structure as the PH domain and act as protein interaction modules via the recognition of polyproline-containing sequences (22,23). However, despite the high structural similarity between SHARPIN and the EVH1 domain of HOMER, interaction of SHARPIN with proline-rich sequences is unlikely because SHARPIN lacks a highly conserved cluster of three surface-exposed aromatic amino acids that form the ligand binding site in EVH1 domains (supplemental Fig. S4B). Indeed, when we tested the ability of SHARPIN to bind peptides derived from classical EVH1 domain ligands using ITC, we could not detect any interaction (data not shown). Furthermore, a yeast two-hybrid screen using the N-terminal 127 amino acids as a bait to identify novel protein partners of the PH domain of SHARPIN did not yield any hits.
Based on these observations, we suggest that in SHARPIN, the PH domain is not used as a protein-ligand interaction platform but rather a scaffold that provides the structural basis for homodimerization that occurs under in vitro conditions as shown by our MALS, AUC, and ITC experiments and in vivo (1). The crystal structure of the SHARPIN PH domain reveals that the dimer is formed primarily by hydrophobic interactions between two helices, which are packed head-to-head in an almost rectangular manner. The interface area covers a solvent accessible surface area of 616 Å 2 (26). A binding site of this magnitude is often characteristic for moderate to weak proteinprotein interactions and therefore in good agreement with the experimental binding constant of 88 and 160 M determined by ITC and AUC, respectively (27). Despite the high K d value determined for PH domain dimerization, the interaction is highly specific. Incorporation of a single electrostatic charge in the center of the hydrophobic framework of the interface by mutating Val-114 to Asp completely abolishes interaction. The fact that SHARPIN dimerization is mediated by a PH domain and not as originally assumed by a predicted coiled coil was unexpected and somewhat surprising. However, in this context, it is interesting to note that dimer formation of proteins mediated by the PH superfold has been suggested before, although the phenomenon has not been investigated on a molecular level. The activity of phosphoinositide-dependent kinase 1 for instance is dependent on 3-phosphoinositide-dependent homodimerization of its C-terminal PH domain (28). Similarly, it has been reported that the PH domain of dynamin undergoes a phosphoinositide binding-coupled monomer-dimer equilibrium (29,30). Although in both of these cases, PH domainmediated dimerization is coupled to the interaction with lipids, it raises the possibility that self-association via PH domains is a more general function for the PH superfold.
In conclusion, the structure of the N-terminal portion of SHARPIN presented here shows that it adopts the PH domain superfold. Our data suggest that, unlike what is generally assumed this domain does not act as a protein interaction mod- ule but as a dimerization domain, thereby extending the manner in which the PH fold can be used as a protein scaffold. Current data suggest that this region does not contribute to the catalytic activity of LUBAC (5), 3 indicating that it may play a role in other physiological functions of SHARPIN that have been described, such as its tumor-associated role or its ability to inhibit ␤1-integrin activation (1,(31)(32)(33). Further studies are required to better understand these functions and to test whether self-association might be a regulated process.