Assembly and structure of Lys33-linked polyubiquitin reveals distinct conformations

Ubiquitylation regulates a multitude of biological processes and this versatility stems from the ability of ubiquitin (Ub) to form topologically different polymers of eight different linkage types. Whereas some linkages have been studied in detail, other linkage types including Lys33-linked polyUb are poorly understood. In the present study, we identify an enzymatic system for the large-scale assembly of Lys33 chains by combining the HECT (homologous to the E6–AP C-terminus) E3 ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1) with linkage selective deubiquitinases (DUBs). Moreover, this first characterization of the chain selectivity of AREL1 indicates its preference for assembling Lys33- and Lys11-linked Ub chains. Intriguingly, the crystal structure of Lys33-linked diUb reveals that it adopts a compact conformation very similar to that observed for Lys11-linked diUb. In contrast, crystallographic analysis of Lys33-linked triUb reveals a more extended conformation. These two distinct conformational states of Lys33-linked polyUb may be selectively recognized by Ub-binding domains (UBD) and enzymes of the Ub system. Importantly, our work provides a method to assemble Lys33-linked polyUb that will allow further characterization of this atypical chain type.


INTRODUCTION
Ubiquitylation is a reversible post-translational modification (PTM) that regulates many cellular processes, including protein degradation, endocytosis, DNA repair and immune response [1]. Addition of ubiquitin (Ub) to a substrate lysine involves a threestep enzymatic cascade involving Ub-activating enzyme (E1), Ub-conjugating enzymes (E2) and Ub-ligating enzymes (E3) [2]. E3 ligases fall into three main classes depending on their mechanism of Ub transfer to substrate. RING (really interesting new gene) ligases transfer Ub directly from the E2 on to substrate whereas HECT (homologous to the E6-AP C-terminus) ligases form a thioester intermediate with Ub before transfer on to substrate lysine [3,4]. RBR (RING-between-RING) ligases form the third class and employ a hybrid mechanism for catalysing Ub transfer [5].
PolyUb chains of eight different linkage types can be formed since the seven lysine residues (Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , Lys 48 and Lys 63 ) and N-terminal Met 1 residue in Ub can themselves accept another molecule of Ub [6]. PolyUb of some linkage types have been structurally characterized and these reveal distinct conformations for different linkages [7]. Crystal structures and solution studies using NMR reveal closed conformations for Lys 6 -, Lys 11 -and Lys 48 -linked diUb as a result of extensive interactions between the Ub moieties [8][9][10][11][12]. In contrast, Lys 63 -and Met 1 -linked diUb adopt extended conformations that lack intermoiety interactions [13]. Interestingly, alternate conformations have been observed for some linkage types, highlighting the flexible nature of polyUb [14,15].
The topologically distinct polyUb linkages are recognized by Ub-binding domain (UBD)-containing proteins to couple ubiquitylation to diverse cellular responses [16]. For instance Lys 48 -linked polyUb target proteins for proteasomal degradation, whereas Lys 63 -and Met 1 -linked polyUb chains have nondegradative roles in DNA damage response and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B-cells) signalling [1,17]. For the remaining linkage types, little is known about their precise cellular function.
Lys 33 chains may not be linked to proteasomal degradation, as the amounts of Lys 33 linkages do not increase following proteasome inhibition [18]. Further, several studies show that Lys 33 chains have non-degradative functions. T-cell antigen receptor (TCR) activation is negatively regulated in a proteolysis independent manner by Lys 33 -linked polyUb, when the RING and HECT E3 ligases, Cbl-b (Casitas B-lineage lymphoma b) and Itch respectively modify the zeta-subunit of the TCR with this Ub chain type [19]. Lys 33 -linkages are also reported to negatively regulate activity of AMPK (AMP-activated protein kinase)-related protein kinases in a non-degradative manner [20]. This linkage type has recently been linked to protein anterograde transport from the trans-Golgi network (TGN), where Lys 33 -linked polyubiquitylation of coronin-7 (Crn7), an F-actin regulator, facilitates its targeting to the TGN, which promotes Factin assembly at TGN and contributes to post-Golgi trafficking [21]. Interestingly, Lys 33 -ubiquitylated Crn7 is recognized by the UBDs of the clathrin adaptor protein Epsin15 to result in translocation of Crn7 to the TGN.
Being a reversible PTM, ubiquitylation is regulated by deubiquitinases (DUBs) that hydrolyse isopeptide bonds between two Ub moieties or between Ub and the targeted protein [6,22]. The DUB TRABID (TRAF-binding domain-containing protein) was recently identified to preferentially hydrolyse Lys 29and Lys 33 -linkages [8,23,24]. Whereas TRABID was shown to regulate Wnt signalling, it is not clear if Lys 33 -linked polyubiquitylation is involved [25]. Whereas these studies point to non-proteolytic roles for Lys 33 linkages in several cellular processes, we have a poor understanding of the ligases that can assemble Lys 33 linkages, the specific signals in response to which they are made, how they are decoded and disassembled.
In the present study, we focused on identifying ligases capable of assembling Lys 33 chains for biochemical and structural characterization. By screening a panel of HECT E3 ligases we identified the uncharacterized ligase AREL1 (apoptosis-resistant E3 Ub protein ligase 1; also known as KIAA0317) to assemble Lys 33 linkages along with other linkages. We then used linkageselective DUBs to remove these additional linkages assembled by AREL1 to obtain pure Lys 33 chains. The enzymatic system we established allowed us to assemble large quantities of Lys 33linked polyUb, thus enabling structural analysis of this atypical chain. We report the first crystal structures of Lys 33 -linked diUb and triUb, which reveal distinct conformations.

cDNA and antibody
All cDNA constructs used in the present study were generated by the DNA cloning team, Division of Signal Transduction Therapy, Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, University of Dundee United Kingdom (Supplementary Table S1). Recombinant proteins and plasmids generated for the present study are available from our reagents website (https://mrcppureagents.dundee.ac.uk/). Anti-Ub antibody was purchased from SIGMA (U5379).

Protein expression and purification
Recombinant GST-fusion proteins were expressed in BL21 Escherichia coli cells. Cultures were grown in 2xTY media to D 600 of 0.6-0.8 and the protein expression was induced by adding 300 μM IPTG and further incubation at 16 • C overnight. Cells were lysed by sonication in lysis buffer [50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 10 % glycerol, 0.075 % 2-mercaptoethanol, 1 mM benzamidine, 1 mM PMSF and complete protease inhibitor cocktail (Roche)]. Bacterial lysate was clarified by centrifugation at 30 000 g for 30 min and incubated subsequently with Glutathione Sepharose 4B resin (GE Healthcare) for 2 h at 4 • C. Resins were washed with high salt buffer (250 mM Tris, pH 7.5, 500 mM NaCl and 5 mM DTT) and low salt buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 10 % glycerol and 1 mM DTT). Purified proteins were eluted in low salt buffer supplemented by 30 mM glutathione or cleaved off from the GST-tag by incubating the beads with C3 protease overnight at 4 • C.

Lys 33 -linked polyUb assembly and purification
Large-scale Lys 33 -linked polyUb chains assembly was carried out in 1.5 ml of reaction volume with 25 mg of Ub (Sigma), 500 nM UBE1, 9 μM UBE2D1, 6.3 μM AREL1, 10 mM ATP, 50 mM Tris/HCl (pH 7.5), 10 mM MgCl 2 and 0.6 mM DTT at 30 • C for 6 h. To remove contaminating linkages, 20 μM Cezanne E 287 K/E 288 K (Cezanne EK), 5 μM OTUB1 and 5 mM DTT were added in to the assembly reaction and incubated further at 30 • C overnight. The reaction mixture was diluted to a total volume of 50 ml of 50 mM sodium acetate (pH 4.5). Lys 33 chains of defined lengths were purified by cation exchange using a Resource S 6 ml column (GE Healthcare), equilibrated in buffer A (50 mM sodium acetate, pH 4.5) and eluted in a gradient with buffer B (50 mM sodium acetate, pH 4.5, 1 M NaCl).

Parallel reaction monitoring MS analysis
PolyUb chains were digested with trypsin and analysed on an LTQ-Velos mass spectrometer (Thermo) fitted with an Easy-Spray Source (Thermo) and utilizing a Dionex RSLC HPLC system. Standard diUb chains were purchased from Boston Biochemicals and a synthetic peptide AK(GG)IQDK representing the tryptic Ub K29 linkage was purchased from Pepceuticals. Digests (prepared in 0.1 % TFA (trifluoroacetic acid)/water) were concentrated on a 20 × 0.1 mm nanotrap column (Thermo) equilibrated in 0.1 % TFA/water (10 μl/min) and washed with 10 μl of the same buffer. The samples were loaded and washed in TFA buffers, as the trap column in the presence of formic acid did not retain the tryptic peptide containing the Lys 29 linkage. Peptides were then separated on a 150 × 0.075 mm PepMap C18, 3 μm Easy-Spray column (Thermo) equilibrated with 2 % acetonitrile/0.1 % formic acid/water at 300 nl/min, employing a stepped gradient of buffer B (80 % acetonitrile/0.1 % formic acid/water) as follows: 0-14 min = 1 %-30 % B, 14-15 min = 30 %-80 % B, 15-20 min = 80 % B. LC-MS data was acquired in data-independent mode with one full scan (m/z = 350-1800) followed by eight product ion scans as described below. Parameters used: Easy-Spray column voltage was 1.9 kV; isolation width was set to 1 Da; normalized collision energy was 35, and the activation time was 10 ms. The ion current for the daughter ions was summed using Xcalibur software (Thermo) for each precursor mass analysed (Supplementary Table  S2). The resultant summed intensities provide the y-axis values for Figure 1(B) and Supplementary Figure S2. This method was more specific than solely using the extracted ion current for the precursor mass for each Ub chain peptide.

Crystallization and structure determination
Purified Lys 33 -linked diUb chains were crystalized at 9 mg/ml in mother liquor containing 200 mM lithium sulfate, 100 mM sodium acetate (pH 4.5) and 50 % PEG400. Further, diffraction quality of the crystal was improved using seeding technique in the presence of 200 mM potassium iodide and 20 % PEG3350 in addition to the mother liquor as mentioned above. Purified Lys 33 -linked triUb chains were crystalized at 8 mg/ml in mother liquor containing 20 mM sodium/potassium phosphate, 100 mM Bis Tris propane (pH 7.5) and 20 % PEG3350. Single crystals obtained from Lys 33 -linked diUb and triUb chains were cryoprotected in the mother liquor containing 20 % and 30 % ethylene glycol respectively. Diffraction data were collected at ESRF (European Synchrotron Radiation Facility) beam line ID29. All data were processed as in described in 'Supplementary Materials and Methods'. Co-ordinates and structure factors for the refined Lys 33 diUb and triUb have been deposited in the Protein Data Bank (PDB, www.rcsb.org) under the accession code 4XYZ and 4Y1H respectively.

Assembly of Lys 33 -linked polyubiquitin
Lys 11 , Lys 48 and Lys 63 chains can be assembled in vitro using E2 enzymes, whereas Lys 6 -and Met 1 -linked polyUb can be assembled by HECT and RBR E3 ligases respectively [9,11,26,27]. Unlike RING E3 ligases, in which the linkage specificity is largely determined by the E2, polyUb assembly by HECT E3 ligases is independent of the inherent linkage preference of the E2 [7]. Therefore, we screened a panel of HECT E3s with the aim of identifying HECT E3 ligases capable of assembling Lys 33 linkages. Either the full-length or the catalytic domains of 16 HECT E3 ligases were expressed as GST fusion proteins in E. coli. We obtained soluble expression for 12 of them, which were then purified to near homogeneity (Supplementary Figure  S1A). We next determined the preferred E2 of a given HECT by comparing ubiquitylation products generated by the HECT in reactions performed with each of the following E2 enzymes: UBE2D1, UBE2D2, UBE2D3 or UBE2L3 ( Figure 1A). Since HECT family ligases interact with UBE2L3 (UbcH7) and the UBE2D (UbcH5) subfamily of E2s, these E2 enzymes were selected for the screens [28,29]. With the exception of UBE3B, KIAA1333 and EDD1, all the tested HECT E3 ligases assembled polyUb chains ( Figure 1A). Further, most of the HECTs work with UBE2D family members ( Figure 1A). In this screen we found that AREL1 assembled shorter polyUb chains compared with the rest of HECT E3s. AREL1 might have slower kinetics as upon prolonged reaction time AREL1 also assembled longer chains (Supplementary Figure S1C). On the other hand, UBE3B, KIAA1333 and EDD1 failed to assemble polyUb chains, even after 6 h incubation ( Figure 1A; Supplementary Figure S1C). Next, we utilized MS to characterize the Ub linkages assembled by the different HECT E3 ligases (Supplementary Figure S1B). We analysed the products of the different HECT-mediated ubiquitylation reactions by parallel reaction monitoring (pRM) LC-MS/MS, a method that exclusively monitors the abundance of the daughter ions belonging to peptides derived from Ub linkages ( Figure 1B) [30]. In accordance with previously published observations, we found that UBE3C assembles Lys 29 and Lys 48 linkages ( Figure 1B) [31]. Further, most of the HECT E3s tested assembled Lys 48 and Lys 63 linkages similar to what had been observed previously [29,32]. Lys 6 linkages are assembled mostly by HUWE1 and Lys 11 linkages are assembled by AREL1 and to some extent by HECW1 and HUWE1 ( Figure 1B). Interestingly, our screen of HECT E3 ligases identified AREL1 as capable of assembling Lys 33 linkages ( Figure 1B).
It is important to note that these in vitro screens assess polyUb linkages assembled by the HECT ligase in the absence of its bona fide substrate. In the presence of physiological substrates, these HECT E3s might assemble different linkages, preferring one linkage type over others. For example, Itch, which assembles Lys 63 linkages in vitro ( Figure 1B), has been reported to modify its substrates with Lys 29 or Lys 48 linkages [33]. Additionally, Itch works co-operatively with RING E3 Cbl-b to ubiquitylate TCRζ with Lys 33 linkages. This suggests that additional factors may influence polyUb assembly by HECT E3 ligases.
Whereas AREL1 makes Lys 33 chains, it also assembles Lys 11 and Lys 48 linkages ( Figures 1B and 2A). To obtain pure Lys 33linked polyUb, the other linkages assembled by AREL1 have to be removed, for which linkage-selective DUBs are required. Cezanne mainly hydrolyses Lys 11 linkages, whereas OTUB1 only cleaves Lys 48 linkages [24]. We used a mutant version of Cezanne (Cezanne EK) that hydrolyses Lys 6 , Lys 11 , Lys 48 and Lys 63 linkages (Supplementary Figure S2). When Cezanne EK and OTUB1 were included in the assembly reaction, the end product was enriched in free polyUb chains and almost 90 % of the input Ub was converted into unanchored or free polyUb chains ( Figure 2B). In order to confirm the linkage type of the resulting polyUb chains, we performed a linkage type analysis using Ub mutants containing lysine-to-arginine substitutions. In the presence of Cezanne EK and OTUB1, free polyUb chain formation was not impaired with K6R, K11R, K27R, K29R, K48R or K63R mutants ( Figure 2C). In contrast, formation of polyUb chains was significantly reduced with the K33R mutant, suggesting that this method generates polyUb chains that are Lys 33 linked ( Figure 2C). Moreover, when incubated with the DUB TRABID that specifically hydrolyses Lys 29 and Lys 33 linkages [23], the assembled polyUb chains were cleaved down to monoUb, confirming the presence of Lys 33 linkages ( Figure 2D). Taken together, these results demonstrate that an Ub chain editing complex made up of the enzymes AREL1, UBE2D1, Cezanne EK and OTUB1 can be used to assemble Lys 33 -linked polyUb chains. We next scaled up the assembly reactions to make a large quantity of Lys 33 -linked chains. Using cation exchange chromatography, we could separate Lys 33 -linked chains of defined lengths containing 2-5 Ub moieties and the purity of Lys 33 -linked diUb and triUb was confirmed by silver staining (Figures 2E  and 2F). pRM LC-MS/MS analyses of purified diUb and triUb validated that the purified polyUb chains produced using this approach only contained Lys 33 linkages and other linkages were not detected (Supplementary Figure S2B). Taken together, these data reveal a robust and reproducible method for generating milligram quantities of Lys 33 -linked polyUb.

Crystal structure of Lys 33 diubiquitin
The topology of polyUb of different linkage types and potentially the length of the polyUb chains determine specificity and outcome of polyUb recognition. We therefore wanted to structurally characterize Lys 33 -linked polyUb chains. We obtained crystals of Lys 33 -linked diUb at 9 mg/ml and the crystals diffracted to 1.65 Å (1 Å = 0.1 nm) resolution. The structure was solved by molecular replacement and refined to the statistics shown in Table 1. The asymmetric unit (ASU) contains one Lys 33 -linked diUb ( Figure 3A). The flexible isopeptide linkage formed between the C-terminus of the distal Ub and Lys 33 of the proximal Ub is not fully resolved in the electron density maps and no clear electron density is present for Gly 76 .
Lys 33 -linked diUb adopts a symmetric compact conformation in the crystal structure with extensive hydrophobic contacts between the proximal and distal moieties. Ile 36 patches of both proximal and distal Ub moieties, which comprise Ile 36 , Leu 71 and Leu 73 , are present at the dimeric interface ( Figure 3B). Further hydrophobic contacts in this symmetric interface involve Leu 8 , Ile 13 and Leu 69 of both moieties ( Figure 3B). Leu 8 is part of a flexible loop in Ub that spans the β1 and β2 strands (β1-β2 loop) and exhibits different conformations in different Ub structures [34]. Depending on the conformation of this loop, Leu 8 is part of either the Ile 36 patch or the orthogonal hydrophobic patch centred on Ile 44 , consisting of residues Ile 44 , Val 70 and His 68 . In the observed Lys 33 diUb structure, this loop is oriented towards Ile 44 and is thus part of the Ile 44 patch ( Figure 3C). In contrast, the β1-β2 loop conformation in the distal Ub of Lys 6 diUb makes Leu 8 part of the Ile 36 patch ( Figure 3C) [8].
In the compact conformation of Lys 6 diUb, the interface is made up of the extended Ile 36 patch from the distal Ub and the Ile 44 patch of the proximal Ub ( Figure 3D). In Lys 48 diUb, the interface is made up of Ile 44 patches of both distal and proximal Ub ( Figure 3D). The compact conformation observed for Lys 33 diUb is distinct from the compact conformations observed for Lys 6 and Lys 48 diUb ( Figure 3D) [8,9,12]. The Ile 36 patches of both distal and proximal Ub in Lys 33 -linked diUb are buried and make up the interface, whereas the Ile 44 patches form a larger hydrophobic surface and are solvent exposed. Molecular modelling approaches predict that Lys 33 -linked diUb exists in an open conformation and cannot adopt a compact conformation due to steric occlusion [35]. However, our crystal structure reveals that Lys 33 -linked diUb can adopt a closed conformation. Intriguingly, the closed conformation of Lys 33 diUb is very similar to that adopted by Lys 11 -linked diUb ( Figure 3D) [10]. Despite the similar conformations adopted by Lys 11 and Lys 33 linkages, DUBs can still distinguish between the two linkage types highlighting the remarkable selectivity present in the Ub system [24].

Crystal structure of Lys 33 triubiquitin
The presence of a symmetric interface raises the question of how chain extension can be achieved and what structure longer Lys 33 polyUb chains adopt. To address this question we purified

Lys-11 Ub2
Distal Proximal  milligram quantities of Lys 33 -linked triUb for crystallization studies. Lys 33 triUb crystallized in a different space group with unit cell dimensions different from that of Lys 33 diUb crystals. Diffraction data were obtained at 1.4 Å resolutions and the structure solved by molecular replacement and refined to the final statistics shown in Table 1. Although we crystallized triUb, the ASU only contains one Ub molecule ( Figures 4A and 4B, chain B). In the crystal lattice, neighbouring Ub molecules complete the trimer where the C-terminus of a symmetry-related molecule (chain C) is close to Lys 33 of chain B; and the C-terminus of chain B is positioned close to Lys 33 residue of chain A ( Figure 4A). Clear electron density is visible for the isopeptide linkage connecting the Ub moieties via Lys 33 (Supplementary Figure S3). In contrast with the compact conformation adopted by Lys 33 diUb that involves extensive hydrophobic interactions at its interface, Lys 33 -linked triUb adopts an open extended conformation. The three Ub molecules of the trimer are arranged in the same orientation forming a linear array, where there are no interactions between the individual Ub moieties apart from the isopeptide linkage ( Figures 4A and 4B). When compared with the compact diUb, the proximal Ub of Lys 33 -linked triUb is rotated by almost 65 • suggesting lack of rotational constraints between individual Ub moieties ( Figure 4C). Further, the hydrophobic patches are exposed to solvent, where symmetric arrangement positions the Ile 44 hydrophobic patches on the same face of the trimer and the Ile 36 patches on another face ( Figure 4D). This extended conformation of Lys 33 chains differs from the fully extended conformations observed for Lys 63 and Met 1 diUb [13]. In the crystal structures of Lys 63 and Met 1 chains, the hydrophobic patches alternate on opposite sides of the chain whereas they are located on the same face in Lys 33 chains (Figures 4D-4F).
Taken together, these results reveal two distinct conformations of Lys 33 -linked Ub chains and the compact and extended conformations observed are distinct from those of Lys 6 , Lys 48 , Met 1 and Lys 63 chains (Supplementary Figure S4). It is to be noted that the diUb structure was obtained from crystals grown at low pH (pH 4.5) whereas the triUb structure was from crystals grown at pH 7.5. This is in contrast with Lys 48 chains that adopt a compact conformation at physiological conditions and an open one at low pH (pH 4.5) [36,37]. The lack of intermoiety interactions in Lys 33 -linked triUb suggests that the relative orientations of the Ub moieties may vary in solution, with our crystal structure representing a snapshot of this dynamic process. Further studies will be required to determine the preferred conformation of Lys 33 chains in solution.
The topology of polyUb chains together with the relative positioning and orientation of the hydrophobic patches are factors that determine linkage selectivity in polyUb binding.  We have determined the structures of Lys 33 -linked diUb and triUb that reveals compact and extended conformations with distinct characteristics. It will be important to analyse how UBDs and DUBs exploit the distinct features of Lys 33 -linked polyUb to achieve linkage-selective recognition. The exposed hydrophobic patches, the unique structural features and the different conformations that can be adopted by Lys 33 -linked polyUb are likely to be exploited by DUBs and UBDs. Further, shorter Lys 33 chains may have different conformations compared with longer chains and this introduces an additional layer of regulation where the length of the polyUb chain may determine which UBD binds and thereby determining the outcome of ubiquitylation.
AREL1 was recently identified as a novel anti-apoptotic E3 Ub ligase [38]. However, the Ub linkages assembled by AREL1 were not investigated. In our in vitro HECT E3 screen, we find that AREL1 mainly assembles Lys 33 and Lys 11 polyUb chains along with small amounts of Lys 48 and Lys 63 linkages. We speculate that AREL1 assembles mixed and branched polyUb chains containing different linkages. There is growing evidence suggesting specialized roles for mixed and branched chains [39,40]. Therefore, it will be important to address whether AREL1 assembles heterotypic chains in cells and what its cellular substrates are. Alternatively, AREL1 may be present in complex with DUBs that could promote ubiquitylation of substrates with homotypic Lys 33 chains. Indeed studying the functional role of AREL1 may reveal insights into the biological roles of Lys 33 -linked polyubiquitylation. Importantly, we provide the first description of an enzymatic system for the large-scale assembly of Lys 33 -linked polyUb, which will pave the way for future studies.