Portal de Periódicos da CAPES

Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation

2007; Springer Nature; Volume: 26; Issue: 11 Linguagem: Inglês

10.1038/sj.emboj.7601711

1460-2075

Puck Knipscheer, Willem J. van Dijk, Jesper V. Olsen, Matthias Mann, Titia K. Sixma,

Glycosylation and Glycoproteins Research

Article10 May 2007free access Noncovalent interaction between Ubc9 and SUMO promotes SUMO chain formation Puck Knipscheer Puck Knipscheer Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands Search for more papers by this author Willem J van Dijk Willem J van Dijk Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands Search for more papers by this author Jesper V Olsen Jesper V Olsen Department of Proteomics and Signaltransduction, Max-Planck Institute for Biochemistry, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Matthias Mann Matthias Mann Department of Proteomics and Signaltransduction, Max-Planck Institute for Biochemistry, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Titia K Sixma Corresponding Author Titia K Sixma Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands Search for more papers by this author Puck Knipscheer Puck Knipscheer Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands Search for more papers by this author Willem J van Dijk Willem J van Dijk Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands Search for more papers by this author Jesper V Olsen Jesper V Olsen Department of Proteomics and Signaltransduction, Max-Planck Institute for Biochemistry, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Matthias Mann Matthias Mann Department of Proteomics and Signaltransduction, Max-Planck Institute for Biochemistry, Am Klopferspitz, Martinsried, Germany Search for more papers by this author Titia K Sixma Corresponding Author Titia K Sixma Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands Search for more papers by this author Author Information Puck Knipscheer1, Willem J van Dijk1, Jesper V Olsen2, Matthias Mann2 and Titia K Sixma 1 1Department of Molecular Carcinogenesis, The Netherlands Cancer Institute and Center for Biomedical Genetics, Plesmanlaan, Amsterdam, The Netherlands 2Department of Proteomics and Signaltransduction, Max-Planck Institute for Biochemistry, Am Klopferspitz, Martinsried, Germany *Corresponding author. Department of Molecular Carcinogenesis, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Tel.: +31 20 5121959; Fax: +31 20 5121954; E-mail: [email protected] The EMBO Journal (2007)26:2797-2807https://doi.org/10.1038/sj.emboj.7601711 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The ubiquitin-related modifier SUMO regulates a wide range of cellular processes by post-translational modification with one, or a chain of SUMO molecules. Sumoylation is achieved by the sequential action of several enzymes in which the E2, Ubc9, transfers SUMO from the E1 to the target mostly with the help of an E3 enzyme. In this process, Ubc9 not only forms a thioester bond with SUMO, but also interacts with SUMO noncovalently. Here, we show that this noncovalent interaction promotes the formation of short SUMO chains on targets such as Sp100 and HDAC4. We present a crystal structure of the noncovalent Ubc9–SUMO1 complex, showing that SUMO is located far from the E2 active site and resembles the noncovalent interaction site for ubiquitin on UbcH5c and Mms2. Structural comparison suggests a model for poly-sumoylation involving a mechanism analogous to Mms2-Ubc13-mediated ubiquitin chain formation. Introduction SUMO is a ubiquitin-related post-translational modifier that plays an important role in many cellular pathways including transcriptional regulation, intracellular transport, DNA repair and replication (Hoege et al, 2002; Pichler and Melchior, 2002; Girdwood et al, 2003; Seeler and Dejean, 2003; Stelter and Ulrich, 2003; Yang et al, 2003; Muller et al, 2004). Sumoylation of substrates generally functions by modulating their interaction properties with other proteins. Although SUMO has been detected mostly as single molecule modification, recent reports show that formation of SUMO chains is also observed for SUMO1 in vitro (Pichler et al, 2002; Pedrioli et al, 2006; Yang et al, 2006) and for SUMO2/3 both in vitro and in vivo (Tatham et al, 2001; Fu et al, 2005). The process of SUMO modification is chemically similar to that of ubiquitin conjugation and the enzymes involved are mostly homologous. This involves the ligation of the C-terminus of the modifier to a lysine residue on the substrate, mediated by a highly regulated three-step cascade. For SUMO, this requires Aos1-Uba2 as E1 or activating enzyme, Ubc9 as E2 or conjugating enzyme and an E3 ligase such as PIAS, Pc2 or RanBP2 (Melchior, 2000; Johnson, 2004), although for many targets the cognate E3 has not yet been identified. In the first step, a thioester bond is formed between the modifier and the catalytic cysteine of the E1 enzyme in an ATP-dependent reaction. This thioester bond is subsequently transferred to the catalytic cysteine of the E2 enzyme, and in the last step the modifier is ligated to the ε-amino group of a lysine on the substrate with or without the help of an E3 ligase. In contrast to ubiquitin conjugation, the E2 enzyme in sumoylation plays an active role in target recognition by interacting with a ΨKxE/D consensus site sequence present on most, but not all targets (Sampson et al, 2001). The process uses one of four vertebrate SUMO isoforms that have partially overlapping target specificity. SUMO2 and SUMO3 differ only by three N-terminal residues and they share 45% sequence identity with SUMO1. The recently identified SUMO4 is more similar to SUMO2/3 (87%) than to SUMO1 (41%). Most of the SUMO1 in cells is found in conjugates, whereas there is a large pool of free cellular SUMO2/3 (Saitoh and Hinchey, 2000; Tatham et al, 2001; Ayaydin and Dasso, 2004). Apparently, only SUMO2/3 form chains on substrates in vivo, whereas SUMO1 chains have only been shown in vitro (Pichler et al, 2002; Pedrioli et al, 2006; Yang et al, 2006). The SUMO2/3 chains are linked through lysine 11, located in a traditional SUMO consensus motif in the flexible N-terminus of SUMO2/3 and seem to play a role in PML localization (Fu et al, 2005). The single yeast SUMO homologue, Smt3, also forms chains in vivo, which are important for the regulation and assembly of the synaptonemal complex during meiosis (Bylebyl et al, 2003; Cheng et al, 2006). Transient interaction is an important feature of the sumoylation process and here the E2, Ubc9, plays a central role by interacting with the E1, SUMO, the E3 and the target at various stages. Structural studies have revealed the nature of interaction of Ubc9 with a target (Bernier-Villamor et al, 2002) and with an E3 enzyme (Reverter and Lima, 2005). Mutational analysis has indicated the interface between Ubc9 and the SUMO E1 to be mainly through its N-terminal helix and the loop between the first and the second β-strand (Bencsath et al, 2002). This would suggest a similar interaction, as was recently shown for another ubiquitin-like molecule, Nedd8, with its E1 APPBP1-UBA3 (Huang et al, 2005). Ubc9 interacts with SUMO both in the thioester intermediate, a complex that has been structurally characterized for several ubiquitin E2s with ubiquitin (Hamilton et al, 2001; McKenna et al, 2003b), as well as in a noncovalent manner. This noncovalent Ubc9–SUMO interaction involves the N-terminal helix of Ubc9, as well as the loop between this helix and the first β-strand, a surface that is also partially used for E1 interaction (Liu et al, 1999; Bencsath et al, 2002; Tatham et al, 2003). As a consequence, SUMO and the E1 can compete directly for interaction with the E2 (Bencsath et al, 2002). The role of the noncovalent interaction between SUMO and Ubc9 is unclear and functional studies have been complicated by this shared interaction site. The noncovalent binding between E2 and modifier is not unique for SUMO, as ubiquitin can also interact noncovalently with some if its E2 enzymes. The details of this interaction were recently shown for ubiquitin bound to UbcH5c (Brzovic et al, 2006) and to the E2 variant enzyme Mms2 (Brzovic et al, 2006; Lewis et al, 2006). The E2 variant Mms2 is thought to position ubiquitin for formation of lysine 63-linked chains of ubiquitin by the E2, Ubc13. The noncovalent interface of UbcH5c is also important for chain formation, but this is thought to follow a different mechanism. Here, we show the crystal structure of the noncovalent complex between SUMO1 and Ubc9. SUMO1 interacts with its β-sheet with a consecutive stretch in Ubc9, connecting the first helix and strand. This site is located distant from the active site cysteine and resembles the Mms2 and UbcH5c ubiquitin noncovalent interfaces. We show that both SUMO1 and SUMO2 interact with Ubc9 similarly. The high-resolution structure enabled us to identify Ubc9 and SUMO mutants that specifically inhibit the interaction between the two proteins. These mutants were used to show that interference with this noncovalent interaction does not affect SUMO thioester formation, but instead strongly reduces SUMO2 chain formation on several targets. A model is presented in which the noncovalent interaction between SUMO and Ubc9 mediates SUMO chain formation, involving a mechanism similar to K63-linked ubiquitin chain formation by the Mms2-Ubc13 heterodimer. Results Crystal structure of noncovalent Ubc9–SUMO complex To get insight in the functional importance of the noncovalent interaction between Ubc9 and SUMO, we solved the crystal structure of this complex using human Ubc9 and SUMO1 lacking the flexible N-terminal 20 amino acids (SUMOΔN20) (Table I). Ubc9–SUMO crystals were grown by mixing the two components in a hanging-drop crystallization setup. The quality of the crystals allowed high-resolution data collection, after which the structure was solved by molecular replacement using the structures of Ubc9 (Tong et al, 1997) and SUMO1 (Pichler et al, 2005) as search models. The crystal structure shows that noncovalent interaction of SUMO1 with Ubc9 occurs on the backside of Ubc9 with respect to the active site cysteine (Figure 1A). Figure 1.Structure of noncovalent Ubc9–SUMO1 complex. (A) Cartoon representation of the Ubc9–SUMO1 crystal structure, Ubc9 in blue and SUMO1 in yellow. The catalytic residue is shown in sticks. (B) Details of the interaction site. Residues of Ubc9 (upper panel) and SUMO1 (lower panel) involved in the interaction shown in sticks, counterpart shown as surface representation. (C) Close-up of Ubc9–SUMO1 interaction. (D) Superposition of the UbcH5c–Ubiquitin complex (purple and green, respectively) and the Ubc9–SUMO1 complex. Only UbcH5c and Ubc9 were used for the superposition, the angle between ubiquitin and SUMO is indicated. (E) Sequence alignment of Ubc9, UbcH5c and Mms2 showing secondary structure elements of Ubc9. Residues that loose at least 20% of their solvent accessible surface area upon complex formation with SUMO/ubiquitin are shown on a yellow background. (F) Sequence alignment of SUMO1, SUMO2 and ubiquitin with secondary structure elements of SUMO1 on top. Residues of SUMO1 involved in Ubc9 interaction (determined as in (E)) are shown on a blue background and homologous residues in SUMO2 are framed. Residues of ubiquitin involved in UbcH5c or Mms2 interaction have a purple background. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Ubc9aSUMO covalent Data collection Space group P21 a, b, c (Å) 49.5, 35.0, 72.9 α, β, γ (°) 90, 93.41, 90 Resolution (Å) 50–1.4 (1.48–1.4)a Rsym 6.0 (22.7) I/σI 6.3 (1.6) Completeness (%) 99.2 (99.3) Redundancy 3.5 (3.1) Refinement Resolution (Å) 50–1.4 Number reflections 49 557 Rwork/Rfree 14.0/17.7 Number of atoms Protein 2130 Ligand/ion 1 Na+ Water 408 B-factors 14.8 Protein 12.5 Ligand/ion 24.6 Water 26.8 r.m.s.d. Bond lengths (Å) 0.014 Bond angles (deg) 1.554 a Highest resolution shell is shown in parenthesis. Complex formation does not cause large conformational changes in either Ubc9 or SUMO. Compared to previous crystal structures the r.m.s.d. is 0.79 Å for SUMO (using the core 77 Cα atoms) and 0.60 Å for Ubc9 (using all Cα atoms). The interface between the two proteins buries 727 Å2 of solvent-accessible surface area on Ubc9 and 642 Å2 on SUMO. This interface is relatively hydrophilic, with five salt bridges, eight direct hydrogen bonds and another 12 hydrogen bonds mediated through defined water molecules, but there are also many van der Waals interactions. On the Ubc9 side, all the residues involved in the interaction are situated in one continuous stretch of sequence at the end of the N-terminal helix, the first β-strand and the intervening loop (Figure 1B and E). This compact region of Ubc9 interacts with three of the five β-strands in SUMO's β-sheet. This is primarily β-strand 5 of SUMO, but also β-strand 1 and 3 and the loops connecting these strands are involved in the interface (Figure 1B and F). Details of the interactions are presented in Figure 1C and Supplementary Figure 1. We compared our Ubc9–SUMO structure with the noncovalent complexes of ubiquitin with the E2 enzyme UbcH5c and the E2 variant Mms2, both determined by NMR (Brzovic et al, 2006; Lewis et al, 2006). Although UbcH5c and Ubc9 are only 36% identical, they both show the E2-specific α/β-fold and superimpose with an r.m.s.d. of 2.5 Å using 136 Cα atoms. The E2 variant Mms2 is only 15% identical to Ubc9 and adopts an E2-like fold lacking the C-terminal helix. It superimposes on Ubc9 with an r.m.s.d. of 1.9 Å using 115 Cα atoms. Secondary structures of ubiquitin and SUMO bound to the E2(-like) proteins are also highly similar, even though their sequence is only 18% conserved (r.m.s.d. 1.4 Å using 75 or 72 Cα atoms for ubiquitin bound to UbcH5c and Mms2, respectively). Roughly, the interaction sites of ubiquitin on UbcH5c and SUMO1 on Ubc9 are conserved, both ubiquitin and SUMO1 interact with the backside of the E2, at least 20 Å away from the active site cysteine, and both use their β-sheet for this interaction. Also, the solvent-accessible surface area buried in the complexes is comparable, such as for Mms2-Ub, this is 641 Å2 and 650 Å2, respectively, and for UbcH5c–Ub, it is 567 Å2 and 556 Å2 (Figure 1E). If we superpose only the E2s, it becomes clear that the relative orientations of the modifiers are slightly different (Figure 1D and E). SUMO1 is rotated 28.4° towards the N-terminal helix of Ubc9, compared to ubiquitin on UbcH5c. The tilting of the ubiquitin in the Mms2 structure is 27.7°, compared to the SUMO, but only 14.4° if we compare it with ubiquitin interacting with UbcH5c (Figure 1D). Although crystal contacts could be involved, we did see the same orientation for SUMO in a second crystal form (data not shown). The difference in the position of the modifiers results in a change of interaction surfaces on the E2s, where SUMO interacts with Ubc9 mainly N-terminally, the ubiquitin interaction surface on UbcH5c and Mms2 is shifted somewhat towards the C-terminus (Figure 1E). SUMO1 and SUMO2 interact with Ubc9 with similar affinities Both SUMO1 and SUMO2 bind noncovalently to Ubc9 (Tatham et al, 2003) and even though they are only 44% identical, the residues in the interface with Ubc9 are relatively well conserved (nine identical, four homologous, three different) (Figure 1F). Of the three nonconserved residues, Gly 81 (Glu 77 in SUMO2) only makes main chain contacts, and the other two, changing Ile 27 into an alanine (Ala 23 in SUMO2) and Val 87 into a threonine (Thr 83 in SUMO2) can be accommodated in the interface without problems. Therefore, we deduce that the interaction mode of Ubc9 with SUMO1 and 2 are likely to be very similar, in agreement with NMR studies of this interface (Liu et al, 1999; Tatham et al, 2003). To determine the affinity of the interaction between Ubc9 and SUMO1 and 2, we used isothermal calorimetric analysis. In isothermal calorimetry (ITC), the absorbance or release of energy of mixing two components that interact with each other can be measured as heat changes. These changes in heat can be used to determine the binding constant and thermodynamic parameters of the interaction. For the interaction between Ubc9 and SUMO1, we determined a Kd of 82±23 nM (Figure 2A). The heat exchange or enthalpy contribution to the binding is relatively small (maximally 6 kcal/mol under these conditions), but high enough to calculate the Kd accurately. Previously, a slightly higher dissociation constant of 250±70 nM has been reported for the Ubc9–SUMO1 interaction using ITC (Tatham et al, 2003). The only obvious differences in Kd measurement between ours and Tatham et al (2003) are a small pH difference (pH=8.0 versus 7.5, respectively) and the presence of an N-terminal His-tag on Ubc9 in their experiments. Both of these factors could contribute to the threefold difference in Kd. When we performed identical ITC measurements replacing SUMO1 for SUMO2, we were not able to measure any reproducible heat exchange during the measurement. Nevertheless, if we collected the sample from the flow cell after the experiment and run it on an analytical gel filtration column, we observe complete complex formation between SUMO2 and Ubc9 (data not shown). As the heat exchange of the reaction for SUMO1 binding was very small, it seems likely that for SUMO2 the enthalpic contribution is even smaller and the reaction is mostly entropically driven, and can therefore not be measured by ITC. Figure 2.SUMO1 and SUMO2 have similar affinity for Ubc9. (A) Isothermal calorimetry data for noncovalent interaction between Ubc9 and SUMO1. Raw (upper panel) and processed (lower panel) data for 7 μM SUMO titrated with 12 μl injections of 70 μM Ubc9. Processed data points were fitted to a model describing a single set of binding sites. Thermodynamic parameters for the interaction are ΔH=−5.96±0.2 kcal/mol and −TΔS=−3.87 kcal/mol (B) Chromatograms of analytical gel filtration runs for Ubc9 with SUMO1 (upper panel) and Ubc9 with SUMO2 (lower panel). Runs of single proteins contained 60 μM Ubc9 or 300 μM SUMO1/2, complex runs contained 50 μM Ubc9 and 100 μM SUMO1/2. Ubc9–SUMO2 chromatogram has been scaled (see second y-axis) because SUMO2 contains few aromatic residues and therefore has low signals. (C) Gel-filtration-based shift assays visualized by Western blot analysis using anti-Ubc9. For SUMO2 (left panel), as well as for SUMO1 (right panel), several gel-filtration runs were performed with a constant Ubc9 and increasing SUMO concentrations (molar ratio is depicted on the left). Seven consecutive fractions ranging in elution volume from 1.15 to 1.4 ml are loaded on the gel for both SUMO1 and SUMO2. Download figure Download PowerPoint For a direct comparison of the affinities of Ubc9 with SUMO1 and SUMO2, we therefore used an analytical gel filtration shift experiment. First, we tested the method using high protein concentrations by mixing pure samples of Ubc9 (50 μM) and an excess of SUMO1 or SUMO2 (100 μM) in 25 μl, incubation at 4 °C for 10 min before running it on a Superdex 75 gel filtration column. Both for SUMO1 (Figure 2B, upper panel) and SUMO2 (Figure 2B, lower panel), all of the Ubc9 were shifted to the Ubc9–SUMO complex peak, whereas the excess of SUMO eluted in a peak overlapping with the free SUMO peak. To compare affinities, we used lower concentrations of Ubc9 (390 nM) with varying SUMO concentrations, followed by gel filtration chromatography and Western blotting of the fractions using a Ubc9 antibody. This allowed visualization of the free Ubc9 peak shifting to the SUMO-bound peak upon increased SUMO concentrations in the samples (Figure 2C). Both SUMO1 and SUMO2 are able to shift the Ubc9 peak under these conditions and the peak shift occurs at similar SUMO1 and SUMO2 concentrations indicating similar affinities of Ubc9 for SUMO1 and SUMO2. Ubc9H20D and SUMO1E67R inhibit noncovalent interaction Although several groups have reported the noncovalent interaction between Ubc9 and SUMO, the function of this interaction has been subject of speculation. Based on mutant analysis, it has been proposed that the interaction is needed for SUMO thioester formation (Tatham et al, 2003). However, as the binding sites for SUMO and the E1 on Ubc9 partially overlap, it was difficult to create interface mutants that do not affect E1 interaction, and consequently, thioester formation. Now, based on our high-resolution structure, we searched for Ubc9 or SUMO1 mutants that only abolish the noncovalent interaction between Ubc9 and SUMO. For SUMO1, we generated the following mutants: E67R, G68Y, V87W and E89R. These mutants were tested for their ability to interact with Ubc9 noncovalently, as well as for their activity in E1 and Ubc9 thioester formation. As summarized in Table II, all of these mutants showed decreased noncovalent interaction with Ubc9 and SUMOE67R, as well as SUMOE89R and SUMOV87W, were completely unable to interact with Ubc9 in the analytical gel filtration assay (Supplementary Figure 2 and data not shown). SUMOE89R and V78W, however, were also impaired in either E1 or Ubc9 thioester formation or both, and were therefore not good candidates to study the function of the Ubc9–SUMO noncovalent interaction (Figure 3A and Supplementary Figure 2). The SUMOE67R mutant only has a minor defect in Ubc9 thioester formation and would, from these mutants, be the best candidate to study the role of noncovalent Ubc9–SUMO interaction (Figure 3B and Supplementary Figure 2). Figure 3.Mutants that interrupt noncovalent Ubc9–SUMO binding but not thioester formation. (A) Thioester formation followed in time for Ubc9 and SUMO1 wild type and mutants. Concentrations were: 100 nM E1, 900 nM Ubc9 and 3 μM SUMO1. (B) Thioester formation assay comparing SUMOWT with SUMOE67R. Concentrations were: 200 nM E1, 1.4 μM Ubc9 and 15 μM SUMO1. (C) Thioester formation assay as in (B) comparing Ubc9WT with Ubc9H20D. (D) Noncovalent binding studied using analytical gel-filtration for Ubc9H20D with SUMO1. Curve indicated as 'Ubc9+SUMO1' was 44 μM Ubc9H20D and 108 μM SUMO1, 'Ubc9+more SUMO1' was 27 μM Ubc9H20D and 136 μM SUMO1. Free Ubc9H20D and the complex between Ubc9WT and SUMO are indicated for clarity. Download figure Download PowerPoint Table 2. Summary of SUMO mutant data SUMO WT E67R G68Y V87W E89R Ubc9 binding ++a − +/− − − E1 thioester ++ ++ +/− ++ − Ubc9 thioester ++ + − +/− − a ++ 90–100% of SUMO WT activity, +70–90% of WT activity, +/− 40–60% of WT activity, − 0–20% of WT activity. For Ubc9, we tested mutations in four residues, R17E, H20D, G23R, and V25W and V25R (Table III). These mutants were tested for SUMO1 interaction and, in addition, for their ability to interact noncovalently with the E1, as well as their activity in Ubc9∼SUMO thioester formation (Figure 3A and Supplementary Figure 2, data not shown). Valine 25 is equivalent to serine 22 in UbcH5c, mutating this residue inhibited noncovalent interaction with ubiquitin (Brzovic et al, 2006). In Ubc9, however, mutation of this residue did not affect the noncovalent binding of SUMO probably due to the fact that it is less well buried in the Ubc9–SUMO interface (Supplementary Figure 2). Also, the G23R mutation did not abolish Ubc9–SUMO noncovalent interaction. In contrast, the R17E and H20D mutants do disturb the interface and are strongly inhibited in Ubc9–SUMO interaction (Figure 3D, Supplementary Figure 2). However, Ubc9R17E was also deficient in E1 interaction and strongly reduced in Ubc9∼SUMO thioester formation and was therefore excluded from further studies. Ubc9H20D was the only Ubc9 mutant that abolished the noncovalent interaction with SUMO (Figure 3D) without affecting thioester formation (Figure 3C), even though it does show a reduction in E1 interaction (Supplementary Figure 2C). This mutation is therefore suited for further analysis of the function of noncovalent interaction between Ubc9 and SUMO. Table 3. Summary of Ubc9 mutant data Ubc9 WT R17E G23R V25W V25R H20D SUMO binding ++a − ++ ++ ++ − E1 interaction ++ − ++ ++ ++ +/− Ubc9 thioester ++ − ND ND ND ++ a As is but for Ubc9 mutants. ND: not determined. Noncovalent Ubc9–SUMO interaction promotes SUMO chain formation In both Mms2 and in UbcH5c, the noncovalent interaction with ubiquitin is involved in ubiquitin chain formation (VanDemark et al, 2001; Brzovic et al, 2006). Therefore, we analyzed whether the noncovalent Ubc9–SUMO interaction was important for free SUMO chain formation. We compared the wild-type and the H20D mutant Ubc9, which has lost the noncovalent interaction. The Ubc9 variants were incubated with SUMO, E1 and ATP at 37°C and SUMO chain formation was followed in time (Figure 4A). Under these conditions, neither SUMO1 nor the SUMO2 K11R mutant, which has lost the SUMO consensus site, forms SUMO chains efficiently, in accordance with previous reports (Tatham et al, 2001). SUMO2WT, however, readily forms chains with Ubc9WT, but the Ubc9H20D mutant is clearly and reproducibly less productive in chain formation (Figure 4A). Thus, loss of the noncovalent interaction inhibits SUMO2 chain formation. Figure 4.Noncovalent Ubc9–SUMO interaction promotes SUMO chain formation. (A) Free SUMO chain formation for SUMO1, SUMO2 and SUMO2K11R with Ubc9WT and Ubc9H20D. Formation of SUMO chains is followed in time using SUMO1 or SUMO2 antibodies as indicated. Concentrations were: 100 nM E1, 400 nM Ubc9 and 20 μM SUMO. Note that Ubc9H20D is also strikingly different in forming higher order Uba2 conjugates that occur as a side effect of the reaction. Uba2 is a known target for sumoylation (Zhao et al, 2004; Hannich et al, 2005). The presence of SUMO-modified Uba2 was confirmed by mass spectrometry (data not shown). (B) Sumoylation of Sp100 with SUMO2 comparing Ubc9WT with Ubc9H20D. Concentrations were: 830 nM GST-Sp100, 175 nM E1, 400 nM Ubc9 and 20 μM SUMO2 and detection was with anti-GST. (C) SUMO chain formation on SP100 with SUMO2, comparing Ubc9WT with Ubc9H20D. Concentrations are identical to (B), except E1 was 150 nM, formation of GST-Sp100∗SUMO2 conjugates is followed in time using either a GST antibody (upper panel) or a SUMO2 antibody (lower panel). (D) SUMO chain formation on Sp100 comparing several mutant proteins. Concentrations were: 1.3 μM GST-Sp100, 10 nM E1, 300 nM Ubc9 and 10 μM SUMO and detection was with anti-GST or anti-SUMO2. (E) Assay as in (C) but using GST-HDAC4 as a target and an HDAC4 antibody for the upper panel. Download figure Download PowerPoint Next, we tested SUMO2 chain formation on a known SUMO target, the transcriptional regulator Sp100. If we stop the reaction after 1 h, we observe mostly monosumoylation of Sp100 and, importantly, this is equally efficient for Ubc9WT compared to Ubc9H20D (Figure 4B). However, at the latest time point in the Ubc9WT reaction, a higher, potentially Sp100∗2 × SUMO, band is appearing. To investigate whether this indicates SUMO chain formation, we performed a similar experiment, but extended the incubation time. At later time points, we now observe several higher bands using Ubc9WT that do not appear if we use Ubc9H20D, shown on Western blots with either GST or SUMO2 antibodies (Figure 4C). These higher order bands are not formed very efficiently and it was important to check whether the higher molecular weight bands are a result of SUMO2 chain formation, or monosumoylation on multiple sites in Sp100. Therefore, we analyzed the Sp100∗SUMO2, Sp100∗2 × SUMO2 and Sp100∗3 × SUMO2 samples by mass spectrometry. In all samples, one major modification site in Sp100 was found at K297, the lysine that has previously been identified as the SUMO modification site (Sternsdorf et al, 1997). In addition, a small fraction (<5%) of a minor site was modified (K387). This indicates that most of the higher order bands are due to chain formation of SUMO2. Mass spectrometric analysis identified primarily SUMO2 K11, and to a small extent K5, as the acceptor lysines on SUMO2 itself. As a control for chain formation, we performed a similar sumoylation reaction using less enzyme, comparing Sp100WT that has lost the SUMO consensus lysine at 297 (Sp100K297R). Under these conditions, we still observe SUMO2 chain formation on Sp100WT, whereas Sp100K297R is hardly modified (Figure 4D). Meanwhile, the consensus site on SUMO2 is also required for the chain formation, as the SUMO2 K11R mutant is also reduced in SUMO2 chain formation (Supplementary Figure 3). These results demonstrate that SUMO chains are formed on Sp100 via the consensus site lysine 297, and that SUMO2 uses primarily lysine 11 to make these chains. To examine the importance of the noncovalent interaction between Ubc9 and SUMO2 for the formation of these chains, we tested the Ubc9H20D mutant in the same Sp100