DOI: 10.1002/cbic.201500011
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A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex Stefanie Sommer,[a] Tobias Ritterhoff,[b] Frauke Melchior,*[b] and Henning D. Mootz*[a] click chemistry. The chemical conjugate proved stable against proteolytic cleavage, in contrast to a Ubc9–SUMO1 isopeptide analogue obtained by auto-SUMOylation. Triazole-linked Ubc9– SUMO1 bound specifically to the preassembled E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9, thus suggesting that it is a suitable thioester mimic. We anticipate interesting prospects for its use as a research tool to study protein complexes involving E2 and E3 enzymes.
Ubiquitin and ubiquitin-like (Ubl) modifiers such as SUMO are conjugated to substrate proteins by E1, E2, and E3 enzymes. In the presence of an E3 ligase, the E2 ~ Ubl thioester intermediate becomes highly activated and is prone to chemical decomposition, thus making biochemical and structural studies difficult. Here we explored a stable chemical conjugate of the E2 enzyme from the SUMO pathway, Ubc9, with its modifier SUMO1 as a structural analogue of the Ubc9 ~ SUMO1 thioester intermediate, by introducing a triazole linkage by biorthogonal
Introduction Post-translational modification of eukaryotic proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers such as SUMO (small ubiquitin-like modifier) is an important mechanism to regulate numerous cellular pathways, including proteasomal degradation, DNA repair, nucleocytoplasmic transport, and signal transduction.[1] Conjugation of Ub and Ubl is catalyzed by an ATP-dependent enzymatic cascade composed of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) to covalently attach the modifier at its carboxy terminus to the e-amino group of an acceptor lysine with an isopeptide bond1 (Scheme 1 A). Demodification of a target is achieved by the activity of Ub- and Ubl-specific isopeptidases.[2] Localization, stability, and activity of target proteins is altered in a highly dynamic manner within the cell as a result of this reversibility. Activation and conjugation of the Ub and Ubl modifiers involve high-energy thioester-bond intermediates (Scheme 1 B). These are formed between the E1 and E2 active-site cysteines and the carboxy terminus of the modifier. The charged E2 is assembled into a complex with a suitable E3 ligase and/or sub-
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Scheme 1. Native and non-native Ub/Ubl–protein linkages. A) Native Ub/Ubl isopeptide linkage. B) Native Ub/Ubl–E2 thioester intermediate. C–D) Nonnative triazole linkages as structural analogues of A) prepared by CuAAC. The native acceptor lysine is replaced by either C) a single cysteine residue followed by further functionalization to an azide (triazole linkage 1), or D) the unnatural amino acid AzF (triazole linkage 2). Note that the Ub/Ubl modifiers lack either both (C) or one (D) of the conserved C-terminal glycine residues.
~ is used for a thioester linkage and indicates chemical lability (re-introduced), * is used for native isopeptide bonds.
strate to enable specific Ub or Ubl discharge. Whereas E3 ligases of the RING type function as adapter molecules that present the Ub/Ubl-E2 adduct in a geometry favoring nucleophilic attack by the target lysine in the substrate,[3] HECT-type enzymes directly participate in catalysis as they bind the modifiers at a catalytic cysteine residue prior to conjugation.[4] However, because of the hydrolytic instability of the E2 thioester linkage, biochemical and structural analysis of protein complexes involving an Ub- or Ubl-loaded E2 enzyme and an E3 ligase (with or without a substrate protein) is challenging.
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[a] Dr. S. Sommer, Prof. Dr. H. D. Mootz Institute of Biochemistry, University of Muenster Wilhelm-Klemm-Strasse 2, 48149 Mnster (Germany) E-mail: [email protected] [b] T. Ritterhoff,+ Prof. Dr. F. Melchior Zentrum fr Molekulare Biologie der Universitt Heidelberg DKFZ-ZMBH Alliance Im Neuenheimer Feld 282, 69120 Heidelberg (Germany) E-mail: [email protected] [+] These authors contributed equally to this work.
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Full Papers zole linkage 2[10a] (Scheme 1 D), the substrate protein can contain cysteine residues, which are not tolerated by some of the other chemical conjugation procedures based on disulfide linkages or thiol-ene chemistry.[11e–g] We reasoned that because of the chemical and proteolytic stability of the triazole linkage, CuAAC-mediated chemical conjugation should also be attractive to generate a structural mimic of a Ub or Ubl thioester bond. We hypothesized that such a stable thioester mimic should prove useful for biochemical experiments, typically precluded by the chemical lability of Ub- and Ubl–E2 thioester intermediates in the presence of E3 ligase. To the best of our knowledge, analogues of catalytically competent Ub- or Ubl–E2 enzymes obtained by CuAAC conjugation or other recent chemical approaches have not been reported. In this study, we applied our CuAAC conjugation approach to replace the reactive thioester in the active site of an E2 conjugating enzyme with a non-hydrolysable triazole analogue. We demonstrate the chemical conjugation of SUMO1 to the E2 enzyme Ubc9 at the catalytic cysteine (C93). This conjugate proved to be stable under reducing conditions as well as in the presence of the multi-subunit RanBP2-SUMO-E3 ligase complex.[13] Furthermore, it displayed proteolytic resistance to SUMO isopeptidase activity, in contrast to a similar thioester surrogate based on an isopeptide linker and obtained by SUMOylation of a Ubc9 (C93K, K101R) mutant. Subsequent interaction studies revealed specific binding of the triazole-linked conjugate to the RanBP2 complex, thus underlining the potential of the stable thioester mimic to study protein complex formation in Ubl conjugation pathways.
To circumvent this problem, several approaches have been applied to generate stable Ub/Ubl–E2 linkages. This involves the generation of a less-reactive oxyester bond by a genetic cysteine-to-serine replacement.[5] Such oxyester intermediates have been used successfully for structural studies investigating the interaction of E3 ligases with charged E2 enzymes;[5b–e] however, the oxyester linkage does not provide complete stability. In the presence of E3 ligases of the HECT type, catalytically inactive E3 mutants can be required.[5b] In the case of RING-type E3 enzymes, the oxyester bond was also shown to undergo hydrolysis, as binding to the RING domain activates the Ub/Ubl–E2 linkage.[5c–e] Furthermore, the preparation of a disulfide-linked Ub–E2 adduct by mutating the last glycine residue of Ub to cysteine has been reported,[6] but this strategy can only be used for E2 enzymes that are free of additional reactive cysteines. Moreover, the instability of the disulfide linkage under reducing conditions limits the applicability of such conjugates. Recently, Ub was enzymatically conjugated with an isopeptide bond to an E2 by replacing the catalytic cysteine with a lysine.[7] This isopeptide-linked Ub–E2 adduct was crystallized in complex with the RING domain of the SUMO-targeted Ub ligase RNF4, thereby obtaining novel insights into the underlying mechanism of preparing the Ub moiety for E3-catalyzed transfer.[7] However, it has to be supposed that the isopeptide linkage will be cleaved in the presence of Ub/Ublspecific isopeptidases. Therefore such conjugates would not be suitable probes for interaction studies in, for example, cellular extracts. There is a related problem in experimentally accessible Ub or Ubl conjugates for the regular linkages of the modifiers to the substrate proteins, although these usually occur with chemically stable (yet proteolytically cleavable) isopeptide bonds. Enzymatic conjugation depends on the substrate specificities of the E2 and E3 enzymes, not all of which can be reconstituted in in vitro assays. Furthermore, they often give rise to conjugate mixtures by modification of more than a single acceptor lysine. We have previously reported[8] a chemical conjugation approach to regioselectively and stoichiometrically attach Ub and SUMO modifiers to target proteins by using CuIcatalyzed azide-alkyne cycloaddition (CuAAC).[9] Subsequent work by us and others has refined and extended this method.[10] In these cases, the acceptor lysine residue in the target protein was either replaced by a unique cysteine, which was then further derivatized to contain an azide functionality, or by an unnatural amino acid that contains the azide in its side chain. The CuAAC reaction with a Ub or SUMO modifier harboring an alkyne functionality at its C terminus then installs a triazole linkage as a structural analogue or mimic of the isopeptide bond (Scheme 1 C and D). The key feature of this technology is freedom over the attachment position of the protein modifier, thus allowing the synthesis of site-specific and homogeneous Ub- and Ubl–protein conjugate analogues. In addition to CuAAC-based chemical conjugation, several approaches using different bioorthogonal or cysteine-related chemistries have been reported.[11] Importantly, the CuAAC methodology is readily compatible with large recombinant proteins, in contrast to fully synthetic approaches.[12] Furthermore, when using tria-
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Results and Discussion Preparation of a triazole93-linked SUMO1–Ubc9 thioester conjugate His6-tagged SUMO1(DG) was C-terminally functionalized with an alkyne group by an aminolysis reaction of an intein-generated SUMO thioester with propargylamine (Pa; Scheme 2). This SUMO construct lacks the terminal glycine residue (replaced by Pa in the modified version). To install the azide, p-azidophenylalanine (AzF) was incorporated in place of the active-site cysteine (C93) of Ubc9 by the nonsense-suppression method.[14] For purification purposes, Ubc9 was N-terminally fused to a streptavidin-binding peptide (SBP) tag. The presence and reactivity of the alkyne and azide functional groups were confirmed by CuAAC reactions with the fluorophores fluoresceinazide (Fl-N3) and dansylamide-alkyne (Figure 1 A, lanes 2 and 3). Incubation of His6-SUMO1(DG)-Pa and SBP-Ubc9(C93AzF) in a 1:2 molar ratio for 30 min under CuAAC conditions then led to the formation of the triazole93-linked protein conjugate (Figure 1 A, lane 4). Densitometric analysis of the gel band intensities revealed an efficiency of about 55 % for this reaction (calculated relative to the amount of the used SUMO1-alkyne reaction partner). After subsequent conjugate purification by affinity chromatography on Ni-NTA and streptactin sepharose, the 2
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Scheme 2. Preparation of a stable Ubc9–SUMO1 conjugate by click chemistry. SUMO1 lacking the last glycine residue (SUMO1(DG)) was prepared as a SUMO1–intein thioester and used for C-terminal modification with propargylamine (Pa). 2-Mercaptoethanesulfonate sodium was added as a thiol catalyst to enable formation of the SUMO–Mesna thioester. Catalytic cysteine 93 of Ubc9 was replaced by p-azidophenylalanine (AzF). Following the CuAAC reaction, the conjugate was purified, and the N-terminal streptavidinbinding peptide (SBP) tag of Ubc9 was removed by TEV protease. The latter step exposed an almost native N terminus of Ubc9 (Met1 replaced with Gly as the only sequence alteration). a) CuSO4, TBTA, TCEP; b) Ni-NTA and streptactin purification; c) TEV digest.
N-terminal SBP-tag of Ubc9 was proteolytically removed by TEV protease digest (Scheme 2 and Figure 1 B).
The triazole93-linked SUMO1(DG)–Ubc9 conjugate specifically binds to the RanBP2–E3 ligase complex
The triazole93-linked SUMO1–Ubc9 conjugate is stable under reducing conditions, and to E3 ligase and isopeptidase activities
Next, we investigated whether the triazole93-linked conjugate represents a suitable surrogate for the native SUMO1–Ubc9 thioester. To this end, interaction studies with the multi-subunit RanBP2–E3 ligase complex[13] were performed. To investigate the binding of the triazole93-linked SUMO1(DG)-Ubc9 conjugate to the RanBP2 complex, conjugate and reconstituted E3 ligase were preincubated on ice overnight then samples were analyzed by size-exclusion chromatography. The triazole conjugate co-eluted with the RanBP2 complex from the column, thus indicating efficient binding to E3 ligase (Figure 3 B, lane 8). Free wild-type (wt) Ubc9,free wt
With the triazole93-linked SUMO1(DG)–Ubc9 conjugate in hand, we wanted to test its behavior under conditions where working with the endogenous thioester is difficult. First, the stability of the conjugate under reducing conditions was tested by incubation in DTT-containing buffer. As a control, a Ubc9 ~ SUMO1 thioester was preformed in vitro. The latter displayed quantitative decomposition, but the triazole conjugate was completely stable in the presence of DTT (Figure 2 A). ChemBioChem 0000, 00, 0 – 0
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Next, we assessed the stability of the triazole conjugate in the presence of a SUMO E3 ligase: the multi-subunit RanBP2–E3 ligase complex.[13] This complex is composed of SUMO1-modified RanGAP1 and Ubc9 that bind synergistically to the RanBP2–E3 ligase region between Ran-binding domains RB3 and RB4 (Figure 3 A). The E3 ligase region itself consists of two 50-amino-acid internal repeats (IR1 and IR2) connected by a short linker (M). Formation of the RanBP2/RanGAP1*SUMO1/Ubc9 complex has been shown to take place at IR1, supported by a SUMO-interacting motif (SIM) at the N terminus of IR1.[13, 15] Of note, the Ubc9 molecule in this complex has only a structural (noncatalytic) function; binding of a second SUMO1-thioester-charged Ubc9 molecule within the IR2 region is required for catalysis[13] (Figure 3 A). As expected, the preformed Ubc9 ~ SUMO1 thioester quickly discharged when incubated with the RanBP2–E3 ligase complex; this gave rise to multi- and or poly-SUMOylated species of the RanBP2 protein. In contrast, the triazole conjugate displayed stability after incubation with the RanBP2 complex (Figure 2 B). Finally, we wanted to test the triazole93-linked SUMO1(DG)–Ubc9 conjugate and an isopeptide-conjugated thioester mimic for suitability under experimental conditions where SUMO isopeptidases might be present. The latter conjugate was prepared in analogy to the recently reported Ub–E2 isopeptide linkage,[7] by mutating the catalytic cysteine of Ubc9 to lysine and the adjacent lysine (101) to arginine (to ensure uniform modification on a single lysine) and subsequent E1-dependent in vitro SUMOylation of the Ubc9(C93K/K101R) construct (data not shown). Both thioester surrogates were incubated with a catalytic fragment of the SUMO isopeptidase SENP1 (SENP1cat, sentrin-specific protease; Figure 2 C). The SUMO1–Ubc9(C93K/K101R) conjugate was slowly cleaved under the applied conditions, but the triazole-linked SUMO1(DG)-Ubc9 conjugate prepared by CuAAC was completely stable in the presence of SENP1cat (Figure 2 C).
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Full Papers azole93-linked SUMO1(DG)–Ubc9 conjugate to this RanBP2 complex mutant was significantly impaired (Figure 3 D, lane 7) compared to the unmutated complex (lane 5). Finally, we compared the binding of the triazole conjugate to RanBP2 complexes with that of the isopeptide-based thioester mimic. These mimics showed very similar behavior towards the E3 ligase complex, as they co-migrated to similar extent with the wt RanBP2 complex and showed similarly reduced interaction with the RanB(IR2mut) complex (Figure 3 D, lanes 6 and 8).
Conclusion We have demonstrated the suitability of CuAACmediated chemical conjugation to generate a chemically, proteolytically, and E3 ligase stable analogue of the highly reactive SUMO1 ~ Ubc9 thioester intermediate formed during enzymatic SUMO conjugation. Alkyne-functionalized SUMO1 and azide-modiFigure 1. Synthesis of a triazole93-linked SUMO1(DG)–Ubc9 thioester conjugate. fied Ubc9 were obtained by recombinant expression A) CuAAC reactions were performed for 30 min at RT and analyzed by SDS-PAGE followed and rapidly conjugated by the CuAAC reaction in by Coomassie staining (left) and UV illumination (right). (*: unidentified by-product.) good yields. The chemical conjugate His6-SUMO1-triB) TEV protease digest of purified His6-SUMO1(DG)-triazole93-SBP-Ubc9 was performed with GST-TEV for 64 h at 4 8C (protease/protein 1:5 (w/w); lanes 1 and 2). The resulting azole93-SBP-Ubc9 could be liberated from an SBP tag His6-SUMO1(DG)-triazole93-Ubc9 was then purified by Ni-NTA chromatography (lane 3: by cleavage with TEV protease and purified. Interacwashing fraction; lanes 4–7: elution fractions). See Scheme 2 for location of the TEV site tion studies with the SUMO-specific RanBP2–E3 ligase in the chemically conjugated protein. complex revealed co-elution of the triazole93-linked conjugate with the complex by size-exclusion chromatography. A single point mutation in the SIM binding groove of the chemically conjugated SUMO1 resulted in SUMO1 and free His6-SUMO1 (negative controls) showed no a significant reduction of this interaction, consistent with the stable interaction with the RanBP2 complex under these condiobservation that SUMOylation reactions on Borealin with the tions (Figure 3 C, lane 3 and 3 B, lanes 5 and 6, respecctively). corresponding SUMO1 mutant are much less efficient than To differentiate between free Ubc9 and the Ubc9 molecule in with wt SUMO1. Importantly, binding of the triazole93-linked the RanBP2 complex, we used a RanBP2 complex containing a C-terminally HA-tagged Ubc9-variant (Figure 3 C). Note that conjugate was also impaired when using a RanBP2 complex the presence of the HA-tag had no effect on the binding propvariant in which the IR2 sequence of RanBP2 was mutated to erties of the triazole conjugate (Figure 3 C, lane 3). eliminate the catalytic center of the E3 ligase. To address the specificity of the interaction with the RanBP2 Taken together, these data suggest that the triazole93-linked complex, we also prepared a triazole93-linked Ubc9 conjugate SUMO1–Ubc9 conjugate functions as a suitable structural mimic for the native SUMO1 ~ Ubc9 thioester. The observed containing a SUMO1(F36L) mutant. F36 participates in SUMO– binding of the chemical SUMO1–Ubc9 conjugate to the preSIM binding,[16] and it has been shown that transfer of this SUMO mutant to the endogenous target protein Borealin in assembled RanBP2/RanGAP1*SUMO1/Ubc9 complex supports RanBP2-complex-mediated SUMOylation reactions is signifiour previous finding that two Ubc9 molecules are required for cantly impaired compared to wt SUMO1.[13] Consistent with RanBP2-complex-dependent SUMOylation:[13] the Ubc9 in the this, reduced RanBP2 complex binding was observed for the complex serves a structural role, and the Ubc9 binding triazole93-linked SUMO1(F36L) (DG)–Ubc9 conjugate (Figure 3 B, a SUMO modifier in thioester linkage is the catalytically important E2 conjugating enzyme. The triazole linkage appears to lane 7). provide sufficiently correct positioning and orientation of To gain further evidence for a specific interaction of the SUMO1 on the Ubc9 scaffold, although it is slightly longer chemical thioester analogue with the E3 ligase complex we than the native thioester linkage (Scheme 1 B and D) and conincluded the RanBP2 (IR2mut) complex variant in our binding tains a different pattern of freely rotatable and conformationalassays (Figure 3 D). This variant contains a RanBP2 fragment ly restricted bonds. In line with this interpretation, binding of with five mutations in the IR2 region (implicated in catalysis in the triazole mimic to the RanBP2 complex was very similar to the assembled E3 ligase complex): a double mutation (I2711A/ that of the isopeptide-based thioester mimic (Figure 3 D). Thus, I2712A) in the putative SIM2 and a triple mutation (L2729A/ chemical conjugation to stably attach SUMO to target proteins L12731A/F2736A) at residues crucial for binding of Ubc9 and by the CuAAC reaction was expanded from mimics of isopepthus for catalytic activity.[13, 15] As expected, binding of the tri-
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Full Papers ampicillin (100 mg mL 1), kanamycin (50 mg mL 1), and chloramphenicol (34 mg mL 1). Synthetic oligonucleotides were purchased from Biolegio (Nijmegen, The Netherlands). Site-directed mutagenesis was performed according to the QuikChange Lightning Site-Directed Mutagenesis protocol (Agilent Technologies). All created plasmids were verified by DNA sequencing (GATC Biotech, Konstanz, Germany, and Seqlab, Gçttingen, Germany). The anti-Ubc9 antibody (sc-10759) was from Santa Cruz Biotechnology (Heidelberg, Germany). For anti-RanBP2, see ref. [18]. Expression plasmids: Plasmid pSS19 (encoding His6-SUMO1(DG)GyrA-CBD) was created from pNW12 (encoding His6-SUFigure 2. Stability of the triazole93-linked SUMO1–Ubc9 conjugate. A) Comparison of the stability of the triazole93MO1(DGG)-Gyr-CBD)[8] by site-dilinked His6-SUMO1(DG)–Ubc9 conjugate and a preformed SUMO1–Ubc9 thioester in the presence of DTT. rected mutagenesis to insert one SUMO1–Ubc9 thioester was preformed with 1.6 mm SUMO E1 enzyme, 8 mm Ubc9, and 24 mm SUMO1 at 30 8C for carboxy-terminal glycine residue 10 min. The preforming reaction was quenched by addition of 60 mm EDTA. Thioester formation showed a yield into SUMO1(DGG) by using oligoof about 20 % (first lane). According to this estimation, 300 nm thioester and His6-SUMO1(DG)-triazole93-Ubc9 nucleotides 5’-GTTTAT CAGGAA were analyzed with non-reducing (50 mm Tris (pH 6.8), 4 m urea, 10 % (v/v) glycerol, and 2 % (w/v) SDS; “ DTT”) GGCTGC ATCACG CAAACG and reducing sample buffer (50 mm Tris (pH 6.8), 2 % (w/v) SDS, 0.1 % (w/v) Bromophenol Blue, 10 % (v/v) glycerol, GGAGAC-3’ (forward, inserted Gly and 100 mm DTT; “+DTT”) by anti-Ubc9 immunoblotting. B) Comparison of the stability of the triazole93-linked codon underlined) and 5’-GTCTCC SUMO1(DG)–Ubc9 conjugate and a preformed SUMO1–Ubc9 thioester towards the RanBP2–E3 ligase complex. SUMO1–Ubc9 thioester was preformed as in A); 300 nm thioester and His6-SUMO1(DG)-triazole93-Ubc9 were incuCGTGAT GCAGCC CGTTTG TTCCTG bated with 50 nm reconstitued RanBP2 complex at 30 8C for 15 min. Samples were analyzed in non-reducing ATAAAC-3’ (reverse). pSS19 was sample buffer by anti-Ubc9 and anti-RanBP2 immunoblotting. (#: excess free Ubc9 from the preforming reaction.) then used a template for a site-diC) Comparison of the stability of the triazole93-linked SUMO1(DG)-Ubc9 conjugate and an isopeptide-linked rected mutagenesis to create the SUMO1-Ubc9(C93K/K101R) conjugate towards SUMO isopeptidase. His6-SUMO1(DG)-triazole93-Ubc9 was incubated F36L mutation into the with various concentrations of SENP1cat (aa 419–644) at 30 8C for 30 min. The isopeptide-linked SUMO1– SUMO1(DG) gene by using oligoUbc9(C93K/K101R) conjugate was treated the same way. Reactions were analyzed by anti-Ubc9 immunoblotting. nucleotides 5’-GATAGC AGTGAG ATACAT CTGAAA GTGAAA ATGACA AC-3’ (forward, L36 codon underlined) and 5’-GTTGTC ATTTTC tide bonds[8, 10] to a mimic for the active-site thioester of an E2 ACTTTC AGATGT ATCTCA CTGCTA TC-3’ (reverse) to create pSS38 enzyme. (His 6-SUMO1(F36L)(DG)-GyrA-CBD). To generate pSS34 (encoding In general, non-hydrolysable Ub/Ubl–E2 thioester mimics pSBP-TEVsite-Ubc9(C93TAG)), a TAG codon was inserted at position prepared by CuAAC provide exciting prospects as novel tools C93 into the Ubc9 open reading frame by site-directed mutageneto enable biochemical and structural characterization of prosis with oligonucleotides 5’-CTTCTG GCACAG TGTAGC TGTCCA tein complexes involving E3 ligases, and potentially even subTTCTAG AGGAAG AC-3’ (forward, TAG codon underlined) and 5’strate proteins. This approach is especially attractive in cases GTCTTC CTCTAG AATGGA CAGCTA CACTG-TGCCAG-AAG-3’ (rewhere conventional strategies to obtain stable thioester verse). From the resulting vector pSS13 (pUbc9C93TAG) the Ubc9C93TAG gene was then amplified by using oligonucleotides mimics are not applicable (e.g., the SUMO E2 enzyme Ubc9, 5’-CCGCTC GAGGAA AACCTG TATTTT CAGGGC TCGGGG ATCGCC for which an oxyester is unstable). The resistance of the triaCTCAGC-3’ (TEV cleavage site underlined, XhoI restriction site in zole linkage (in contrast to the isopeptide linkage) towards isoitalics) and 5’-CCCAAG CTTTTA TGAGGG GGCAAA C-3’ (HindIII repeptidase activity will also enable the use of these mimics for striction site in italics). The SBP gene was amplified from pSS02 interaction studies in cell lysate without the need to inactivate (pSBP-HA-GpD-PML11(TAG))[10a] by using oligonucleotides 5’-CATGCC isopeptidases by harsh treatments, such as exposure to high ATGGAC GAAAAA ACCACC-3’ (forward, NcoI site in italics) and 5’concentration of N-ethylmaleimide (NEM). Such experiments CCGCTC GAGGCC GGGTTC ACGCTG AC-3’ (reverse, XhoI site in italmight help to identify previously unknown E3 ligases or other ics). The PCR products were digested with the appropriate enzymes and ligated simultaneously into an NcoI/HindIII digested cellular components interacting with Ub- or Ubl-loaded E2 enpET28a-vector to give pSS34. Plasmids for RanBP2RB3 4 (aa 2304– zymes. 3062), hRanGAP1, wt SUMO1, wt Ubc9, and GST-SENP1cat (aa 419– 644) have been described previously.[13, 17]
Experimental Section
Protein production and purification: For the expression of His6SUMO1(DG)-GyrA-CBD and His6-SUMO1(F36L)(DG)-GyrA-CBD, Escherichia coli BL21(DE3) cells were transformed with the respective
General: Standard molecular biology protocols were applied for DNA cloning and expression. The antibiotics were
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Full Papers EDTA (1 mm)). After washing with buffer W, the protein was eluted with buffer E (Tris (100 mm, pH 8.0), NaCl (150 mm), EDTA (1 mm), desthiobiotin (2 mm)) followed by dialysis in dialysis buffer (HEPES (20 mm, pH 8.0), NaCl (150 mm)). In vitro reconstitution of the RanBP2 complex (His6-RanBP2RB3 4 fragment (aa 2304–3062), fulllength untagged RanGAP1*SUMO1, and full-length Ubc9) was performed as previously reported.[13] CuAAC and purification of conjuSUMO1-triazole93-Ubc9 gates: Reactions were performed in dialysis buffer. SBP-TEVsiteUbc9(C93AzF) (20 mm) and Pamodified SUMO1(DG) (10 mm) were mixed and treated with Tris(2-carboxyethyl)phosphine (TCEP; 500 mm). Then Tris(benzyltriazolyl-methyl)amine (TBTA, 50 mm; in DMSO/tBuOH (1:4)) and CuSO4 (500 mm) were added. After incubation for 30 min at room temperature, the reaction was stopped by addition of EDTA (10 mm). Control reactions with Figure 3. The triazole93-linked SUMO1-Ubc9 thioester analogue specifically binds to the RanBP2–SUMO E3 ligase the fluorophores N-(3-azidopropcomplex. A) Schematic representation of the multi-subunit RanBP2/RanGAP1*SUMO1/Ubc9 E3 ligase complex. The yl)-fluorescein-amide (CLK-FA005-1, complex was in vitro reconstituted from the indicated RanBP2RB3 4 fragment (aa 2304–3062: Ran-binding sites Jena Bioscience, Jena, Germany) RB3 and RB4 and the E3 ligase domain (IR1-M-IR2)). A putative SIM (SIM2) is at the N terminus of IR2. B) Gel filtration interactions studies. The RanBP2 complex was preincubated with triazole93-linked conjugates and the indicatand dansylamide-alkyne[8] (both ed SUMO1 variants (2 mm) on ice overnight, then samples were run on a Superdex 200 column. Fractions at 0.42 dissolved in DMF) were performed column volume were analyzed with Coomassie-stained gels. C) Binding study as above for a RanBP2 complex similarly, but in the dark. All reacwith C-terminally HA-tagged Ubc9, the triazole93-linked conjugate, and Ubc9. D) Binding study as above for the tions were analyzed by SDS-PAGE RanBP2 (IR2mut) complex variant and the isopeptide-linked SUMO1–Ubc9(C93K/K101R) conjugate, as well as the with UV illumination. The efficiency triazole93-linked conjugate. (RanBP2 (IR2mut) = I2711A/I2712A/L12729A/L12731A/F2736A; *: ovalbumin in the of the CuAAC reaction was deterbuffer.) mined by densitometric analysis of band intensities of a Coomassiestained SDS-PAGE gel with Scion Image (Scion Corporation) plasmids and cultured in LB medium supplemented with ampicillin software. Percentage of triazole conjugate formation was calculatat 37 8C to OD600 = 0.6, then protein expression was induced with ed relative to the amount of the used SUMO1-alkyne reaction partIPTG (0.4 mm) for 4 h at 28 8C. To incorporate p-azidophenylalanine ner. (AzF), E. coli BL21 (DE3) cells were co-transformed with the plasmid encoding SBP-TEVsite-Ubc9(C93TAG) and the plasmid providing the To purify His6-SUMO1(DG)-triazole93-SBP-TEVsite-Ubc9 conjugates, genetic information for the orthogonal AzFRS/AzFtRNACUA pair deCuAAC reaction mixtures were dialyzed against buffer W supplerived from Methanococcus jannashii (kindly provided by Peter G. mented with DTT (2 mm). After purification over streptactin seSchultz, Scripps Institute, La Jolla, CA). Cells were grown in M9 minpharose (as above), pooled elution fractions were dialyzed against imal medium (Na2HPO4 (45 mm), KH2PO4 (25 mm), NaCl (8.5 mm), Ni-NTA buffer A (Tris (50 mm, pH 8.0), NaCl (300 mm)) with b-merMgSO4 (1 mm), CaCl2 (0.1 mm), FeCl3 (22 nm), thiamine captoethanol (2 mm) and immobilized on Ni-NTA sepharose 1 (0.03 mg mL ), NH4Cl (0.1 %), glucose (0.2 %)) supplemented with (5Prime, Hamburg, Germany). After washing with buffer A supplekanamycin and chloramphenicol to OD600 = 0.6. AzF (1 mm; mented with imidazole (20 mm then 40 mm), elution was perBachem, Weil am Rhein, Germany) was added, and protein expresformed by addition of buffer A supplemented with imidazole sion was induced with IPTG (1 mm) for 4 h at 37 8C. (250 mm). To cleave the N-terminal SBP-sequence of Ubc9, purified Cells were lysed by two passages through a C5 emulsifier (Avestin, conjugates were mixed with GST-TEV (protein/protease 5:1, w/w) Ottawa, ON). Purification of CBD-tagged SUMO proteins and subseand dialyzed against TEV cleavage buffer (Tris (50 mm, pH 8.0), quent C-terminal modification with propargylamine (Pa) were perEDTA (0.5 mm), DTT (1 mm)) at 4 8C for 64 h to allow complete formed as previously described.[8, 10a] SBP-TEVsite-Ubc9(C93AzF) was conversion. To remove free SBP and the GST-TEV, the His6immobilized on streptactin sepharose (IBA, Gçttingen, Germany) SUMO1(DG)-triazole93-Ubc9 conjugate was immobilized on Ni-NTA sepharose and finally dialyzed against assay buffer (HEPES-KOH equilibrated in buffer W (Tris (100 mm, pH 8.0), NaCl (150 mm),
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[2] a) D. Komander, M. J. Clague, S. Urb, Nat. Rev. Mol. Cell Biol. 2009, 10, 550 – 563; b) C. M. Hickey, N. R. Wilson, M. Hochstrasser, Nat. Rev. Mol. Cell Biol. 2012, 13, 755 – 766. [3] R. J. Deshaies, C. A. P. Joazeiro, Annu. Rev. Biochem. 2009, 78, 399 – 434. [4] D. Rotin, S. Kumar, Nat. Rev. Mol. Cell Biol. 2009, 10, 398 – 409. [5] a) M. L. Sullivan, R. D. Vierstra, J. Biol. Chem. 1993, 268, 8777 – 8780; b) H. B. Kamadurai, J. Souphron, D. C. Scott, D. M. Duda, D. J. Miller, D. Stringer, R. C. Piper, B. A. Schulman, Mol. Cell 2009, 36, 1095 – 1102; c) A. Plechanovov, E. G. Jaffray, S. A. McMahon, K. A. Johnson, I. Navrtilov, J. H. Naismith, R. T. Hay, Nat. Struct. Mol. Biol. 2011, 18, 1052 – 1059; d) J. N. Pruneda, P. J. Littlefield, S. E. Soss, K. A. Nordquist, W. J. Chazin, P. S. Brzovic, R. E. Klevit, Mol. Cell 2012, 47, 933 – 942; e) H. Dou, L. Buetow, G. J. Sibbet, K. Cameron, D. T. Huang, Nat. Struct. Mol. Biol. 2012, 19, 876 – 883. [6] N. Merkley, K. R. Barber, G. S. Shaw, J. Biol. Chem. 2005, 280, 31732 – 31738. [7] A. Plechanovov, E. G. Jaffray, M. H. Tatham, J. H. Naismith, R. T. Hay, Nature 2012, 489, 115 – 120. [8] N. D. Weikart, H. D. Mootz, ChemBioChem 2010, 11, 774 – 777. [9] a) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057 – 3064; b) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41, 2596 – 2599; Angew. Chem. 2002, 114, 2708 – 2711. [10] a) S. Sommer, N. D. Weikart, A. Brockmeyer, P. Janning, H. D. Mootz, Angew. Chem. Int. Ed. 2011, 50, 9888 – 9892; Angew. Chem. 2011, 123, 10062 – 10066; b) N. D. Weikart, S. Sommer, H. D. Mootz, Chem. Commun. 2012, 48, 296 – 298; c) T. Dresselhaus, N. D. Weikart, H. D. Mootz, M. P. Waller, RSC Adv. 2013, 3, 16122 – 16129; d) N. D. van Treel, H. D. Mootz, J. Pept. Sci. 2014, 20, 121 – 127; e) S. Eger, M. Scheffner, A. Marx, M. Rubini, J. Am. Chem. Soc. 2010, 132, 16337 – 16339; f) D. Schneider, T. Schneider, D. Rçsner, S. Malhotra, F. Mortensen, T. U. Mayer, M. Scheffner, A. Marx, Bioorg. Med. Chem. 2013, 21, 3430 – 3435; g) T. Schneider, D. Schneider, D. Rçsner, S. Malhotra, F. Mortensen, T. U. Mayer, M. Scheffner, A. Marx, Angew. Chem. Int. Ed. 2014, 53, 12925 – 12929; Angew. Chem. 2014, 126, 13139 – 13143. [11] a) L. Yin, B. Krantz, N. S. Russell, S. Deshpande, K. D. Wilkinson, Biochemistry 2000, 39, 10001 – 10010; b) X. Li, T. Fekner, J. J. Ottesen, M. K. Chan, Angew. Chem. Int. Ed. 2009, 48, 9184 – 9187; Angew. Chem. 2009, 121, 9348 – 9351; c) S. Virdee, Y. Ye, D. P. Nguyen, D. Komander, J. W. Chin, Nat. Chem. Biol. 2010, 6, 750 – 757; d) A. Shanmugham, A. Fish, M. P. A. Luna-Vargas, A. C. Faesen, F. El Oualid, T. K. Sixma, H. Ovaa, J. Am. Chem. Soc. 2010, 132, 8834 – 8835; e) C. Chatterjee, R. K. McGinty, B. Fierz, T. W. Muir, Nat. Chem. Biol. 2010, 6, 267 – 269; f) J. Chen, Y. Ai, J. Wang, L. Haracska, Z. Zhuang, Nat. Chem. Biol. 2010, 6, 270 – 272; g) E. M. Valkevich, R. G. Guenette, N. A. Sanchez, Y.-c. Chen, Y. Ge, E. R. Strieter, J. Am. Chem. Soc. 2012, 134, 6916 – 6919; h) H. P. Hemantha, S. N. Bavikar, Y. Herman-Bachinsky, N. Haj-Yahya, S. Bondalapati, A. Ciechanover, A. Brik, J. Am. Chem. Soc. 2014, 136, 2665 – 2673. [12] a) C. Chatterjee, R. K. McGinty, J.-P. Pellois, T. W. Muir, Angew. Chem. Int. Ed. 2007, 46, 2814 – 2818; Angew. Chem. 2007, 119, 2872 – 2876; b) K. S. A. Kumar, L. Spasser, L. A. Erlich, S. N. Bavikar, A. Brik, Angew. Chem. Int. Ed. 2010, 49, 9126 – 9131; Angew. Chem. 2010, 122, 9312 – 9317; c) F. El Oualid, R. Merkx, R. Ekkebus, D. S. Hameed, J. J. Smit, A. de Jong, H. Hilkmann, T. K. Sixma, H. Ovaa, Angew. Chem. Int. Ed. 2010, 49, 10149 – 10153; Angew. Chem. 2010, 122, 10347 – 10351; d) M. Hejjaoui, M. Haj-Yahya, K. S. A. Kumar, A. Brik, H. A. Lashuel, Angew. Chem. Int. Ed. 2011, 50, 405 – 409; Angew. Chem. 2011, 123, 425 – 429. [13] A. Werner, A. Flotho, F. Melchior, Mol. Cell 2012, 46, 287 – 298. [14] J. W. Chin, S. W. Santoro, A. B. Martin, D. S. King, L. Wang, P. G. Schultz, J. Am. Chem. Soc. 2002, 124, 9026 – 9027. [15] D. Reverter, C. D. Lima, Nature 2005, 435, 687 – 692. [16] O. Kerscher, EMBO Rep. 2007, 8, 550 – 555. [17] a) A. Pichler, A. Gast, J. S. Seeler, A. Dejean, F. Melchior, Cell 2002, 108, 109 – 120; b) G. Bossis, F. Melchior, Mol. Cell 2006, 21, 349 – 357. [18] S. Hutten, A. Flotho, F. Melchior, R. H. Kehlenbach, Mol. Biol. Cell 2008, 19, 2300 – 2310.
Preparation of isopeptide93-linked SUMO1-Ubc9-conjugate: For formation of the isopeptide-linked conjugate, C93 (catalytic cysteine of Ubc9) was mutated to lysine. In order to achieve specific modification at residue 93, Lys101 was additionally mutated to arginine. The isopeptide-linked conjugate was formed by a SUMOylation reaction with a mixture of Ubc9 (C93K/K101R) (50 mm), SUMO1 (50 mm), SUMO E1 enzyme (500 nm; both prepared in the lab), and ATP (5 mm) in mimic buffer (Tris-HCl (50 mm, pH 10.0), NaCl (150 mm), MgCl2 (5 mm), DTT (1 mm), aprotinin, leupeptin, pepstatin (1 mg mL 1 each; Biolmol)). The reaction was incubated for 18–24 h at 30 8C. The mimic was subsequently purified by size exclusion chromatography on a Superdex75 GL gel filtration column equilibrated in assay buffer supplemented with aprotinin, leupeptin, pepstatin (1 mg mL 1 each). In vitro formation of a SUMO1–Ubc9 thioester: SUMO1–Ubc9 thioester was formed with SUMO E1 enzyme (1.6 mm), Ubc9 (8 mm), and SUMO1 (24 mm) in assay buffer (but with 0.1 mm DTT not 1 mm) supplemented with ovalbumin (0.2 mg mL 1), Tween 20 (0.05 %, v/v), and aprotinin, leupeptin, pepstatin (1 mg mL 1 each) at 30 8C for 10 min. The reaction was quenched by addition of EDTA (60 mm). E3 ligase stability assay: Triazole93-linked SUMO1(DG)-Ubc9 (300 nm) and preformed SUMO1–Ubc9 thioester (300 nm) were incubated with RanBP2 complex (50 nm) in the above buffer for 15 min at 30 8C. Samples were analyzed by immunoblotting with anti-Ubc9 and anti-RanBP2 antibodies. SENP1 assay: Triazole93-linked SUMO1(DG)-Ubc9 (1 mm) and SUMO1-Ubc9(C93K/K101R) (1 mm) were incubated in assay buffer supplemented with ovalbumin (0.2 mg mL 1), Tween 20 (0.05 %, v/ v), and aprotinin, leupeptin, pepstatin (1 mg mL 1 each) for 30 min at 30 8C with different concentrations of GST-SENP1cat (0, 2, 20, or 200 nm). Samples were analyzed by anti-Ubc9 immunoblotting. Gel filtration binding assays with the RanBP2–E3 ligase complex: RanBP2 complexes were incubated with triazole93-linked SUMO1(DG)-Ubc9 conjugate (2 mm), Ubc9 wt (2 mm), and SUMO1 wt (2 mm) in assay buffer supplemented with ovalbumin (0.2 mg mL 1), Tween 20 (0.05 %, v/v), and aprotinin, leupeptin, pepstatin (1 mg mL 1 each) on ice overnight. Samples were applied on a Superdex 200 5/150 GL gel filtration column equilibrated in assay buffer supplemented with aprotinin, leupeptin, pepstatin (1 mg mL 1 each). Fractions at 0.42 column volume were analyzed by Coomassie-stained gels.
Acknowledgements We thank Nadine D. van Treel for discussions and Peter G. Schultz (The Scripps Research Institute) for providing plasmids. H.D.M. and F.M. acknowledge funding by the Deutsche Forschungsgemeinschaft (SFB858, GRK1188 and SFB638, respectively). Keywords: biological activity · click chemistry · enzyme catalysis · post-translational modifications · SUMO · ubiquitin [1] a) A. Hershko, A. Ciechanover, Annu. Rev. Biochem. 1998, 67, 425 – 479; b) O. Kerscher, R. Felberbaum, M. Hochstrasser, Annu. Rev. Biochem. 2006, 22, 159 – 180; c) A. Flotho, F. Melchior, Annu. Rev. Biochem. 2013, 82, 357 – 385.
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FULL PAPERS S. Sommer, T. Ritterhoff, F. Melchior,* H. D. Mootz*
Chemical camouflage: Ubiquitin and SUMO bound as thioesters to their E2 conjugating enzymes are labile intermediates in the presence of an E3 ligase, a fact that hampers biochemical characterization of the charged E2/E3 complexes. A stable chemical conjugate of SUMO1 with the SUMO E2 enzyme is now reported as a thioester mimic to address these limitations.
&& – && A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex
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A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex Stefanie Sommer,[a] Tobias Ritterhoff,[b] Frauke Melchior,*[b] and Henning D. Mootz*[a] click chemistry. The chemical conjugate proved stable against proteolytic cleavage, in contrast to a Ubc9–SUMO1 isopeptide analogue obtained by auto-SUMOylation. Triazole-linked Ubc9– SUMO1 bound specifically to the preassembled E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9, thus suggesting that it is a suitable thioester mimic. We anticipate interesting prospects for its use as a research tool to study protein complexes involving E2 and E3 enzymes.
Ubiquitin and ubiquitin-like (Ubl) modifiers such as SUMO are conjugated to substrate proteins by E1, E2, and E3 enzymes. In the presence of an E3 ligase, the E2 ~ Ubl thioester intermediate becomes highly activated and is prone to chemical decomposition, thus making biochemical and structural studies difficult. Here we explored a stable chemical conjugate of the E2 enzyme from the SUMO pathway, Ubc9, with its modifier SUMO1 as a structural analogue of the Ubc9 ~ SUMO1 thioester intermediate, by introducing a triazole linkage by biorthogonal
Introduction Post-translational modification of eukaryotic proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers such as SUMO (small ubiquitin-like modifier) is an important mechanism to regulate numerous cellular pathways, including proteasomal degradation, DNA repair, nucleocytoplasmic transport, and signal transduction.[1] Conjugation of Ub and Ubl is catalyzed by an ATP-dependent enzymatic cascade composed of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3) to covalently attach the modifier at its carboxy terminus to the e-amino group of an acceptor lysine with an isopeptide bond1 (Scheme 1 A). Demodification of a target is achieved by the activity of Ub- and Ubl-specific isopeptidases.[2] Localization, stability, and activity of target proteins is altered in a highly dynamic manner within the cell as a result of this reversibility. Activation and conjugation of the Ub and Ubl modifiers involve high-energy thioester-bond intermediates (Scheme 1 B). These are formed between the E1 and E2 active-site cysteines and the carboxy terminus of the modifier. The charged E2 is assembled into a complex with a suitable E3 ligase and/or sub-
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Scheme 1. Native and non-native Ub/Ubl–protein linkages. A) Native Ub/Ubl isopeptide linkage. B) Native Ub/Ubl–E2 thioester intermediate. C–D) Nonnative triazole linkages as structural analogues of A) prepared by CuAAC. The native acceptor lysine is replaced by either C) a single cysteine residue followed by further functionalization to an azide (triazole linkage 1), or D) the unnatural amino acid AzF (triazole linkage 2). Note that the Ub/Ubl modifiers lack either both (C) or one (D) of the conserved C-terminal glycine residues.
~ is used for a thioester linkage and indicates chemical lability (re-introduced), * is used for native isopeptide bonds.
strate to enable specific Ub or Ubl discharge. Whereas E3 ligases of the RING type function as adapter molecules that present the Ub/Ubl-E2 adduct in a geometry favoring nucleophilic attack by the target lysine in the substrate,[3] HECT-type enzymes directly participate in catalysis as they bind the modifiers at a catalytic cysteine residue prior to conjugation.[4] However, because of the hydrolytic instability of the E2 thioester linkage, biochemical and structural analysis of protein complexes involving an Ub- or Ubl-loaded E2 enzyme and an E3 ligase (with or without a substrate protein) is challenging.
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[a] Dr. S. Sommer, Prof. Dr. H. D. Mootz Institute of Biochemistry, University of Muenster Wilhelm-Klemm-Strasse 2, 48149 Mnster (Germany) E-mail: [email protected] [b] T. Ritterhoff,+ Prof. Dr. F. Melchior Zentrum fr Molekulare Biologie der Universitt Heidelberg DKFZ-ZMBH Alliance Im Neuenheimer Feld 282, 69120 Heidelberg (Germany) E-mail: [email protected] [+] These authors contributed equally to this work.
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Full Papers zole linkage 2[10a] (Scheme 1 D), the substrate protein can contain cysteine residues, which are not tolerated by some of the other chemical conjugation procedures based on disulfide linkages or thiol-ene chemistry.[11e–g] We reasoned that because of the chemical and proteolytic stability of the triazole linkage, CuAAC-mediated chemical conjugation should also be attractive to generate a structural mimic of a Ub or Ubl thioester bond. We hypothesized that such a stable thioester mimic should prove useful for biochemical experiments, typically precluded by the chemical lability of Ub- and Ubl–E2 thioester intermediates in the presence of E3 ligase. To the best of our knowledge, analogues of catalytically competent Ub- or Ubl–E2 enzymes obtained by CuAAC conjugation or other recent chemical approaches have not been reported. In this study, we applied our CuAAC conjugation approach to replace the reactive thioester in the active site of an E2 conjugating enzyme with a non-hydrolysable triazole analogue. We demonstrate the chemical conjugation of SUMO1 to the E2 enzyme Ubc9 at the catalytic cysteine (C93). This conjugate proved to be stable under reducing conditions as well as in the presence of the multi-subunit RanBP2-SUMO-E3 ligase complex.[13] Furthermore, it displayed proteolytic resistance to SUMO isopeptidase activity, in contrast to a similar thioester surrogate based on an isopeptide linker and obtained by SUMOylation of a Ubc9 (C93K, K101R) mutant. Subsequent interaction studies revealed specific binding of the triazole-linked conjugate to the RanBP2 complex, thus underlining the potential of the stable thioester mimic to study protein complex formation in Ubl conjugation pathways.
To circumvent this problem, several approaches have been applied to generate stable Ub/Ubl–E2 linkages. This involves the generation of a less-reactive oxyester bond by a genetic cysteine-to-serine replacement.[5] Such oxyester intermediates have been used successfully for structural studies investigating the interaction of E3 ligases with charged E2 enzymes;[5b–e] however, the oxyester linkage does not provide complete stability. In the presence of E3 ligases of the HECT type, catalytically inactive E3 mutants can be required.[5b] In the case of RING-type E3 enzymes, the oxyester bond was also shown to undergo hydrolysis, as binding to the RING domain activates the Ub/Ubl–E2 linkage.[5c–e] Furthermore, the preparation of a disulfide-linked Ub–E2 adduct by mutating the last glycine residue of Ub to cysteine has been reported,[6] but this strategy can only be used for E2 enzymes that are free of additional reactive cysteines. Moreover, the instability of the disulfide linkage under reducing conditions limits the applicability of such conjugates. Recently, Ub was enzymatically conjugated with an isopeptide bond to an E2 by replacing the catalytic cysteine with a lysine.[7] This isopeptide-linked Ub–E2 adduct was crystallized in complex with the RING domain of the SUMO-targeted Ub ligase RNF4, thereby obtaining novel insights into the underlying mechanism of preparing the Ub moiety for E3-catalyzed transfer.[7] However, it has to be supposed that the isopeptide linkage will be cleaved in the presence of Ub/Ublspecific isopeptidases. Therefore such conjugates would not be suitable probes for interaction studies in, for example, cellular extracts. There is a related problem in experimentally accessible Ub or Ubl conjugates for the regular linkages of the modifiers to the substrate proteins, although these usually occur with chemically stable (yet proteolytically cleavable) isopeptide bonds. Enzymatic conjugation depends on the substrate specificities of the E2 and E3 enzymes, not all of which can be reconstituted in in vitro assays. Furthermore, they often give rise to conjugate mixtures by modification of more than a single acceptor lysine. We have previously reported[8] a chemical conjugation approach to regioselectively and stoichiometrically attach Ub and SUMO modifiers to target proteins by using CuIcatalyzed azide-alkyne cycloaddition (CuAAC).[9] Subsequent work by us and others has refined and extended this method.[10] In these cases, the acceptor lysine residue in the target protein was either replaced by a unique cysteine, which was then further derivatized to contain an azide functionality, or by an unnatural amino acid that contains the azide in its side chain. The CuAAC reaction with a Ub or SUMO modifier harboring an alkyne functionality at its C terminus then installs a triazole linkage as a structural analogue or mimic of the isopeptide bond (Scheme 1 C and D). The key feature of this technology is freedom over the attachment position of the protein modifier, thus allowing the synthesis of site-specific and homogeneous Ub- and Ubl–protein conjugate analogues. In addition to CuAAC-based chemical conjugation, several approaches using different bioorthogonal or cysteine-related chemistries have been reported.[11] Importantly, the CuAAC methodology is readily compatible with large recombinant proteins, in contrast to fully synthetic approaches.[12] Furthermore, when using tria-
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Results and Discussion Preparation of a triazole93-linked SUMO1–Ubc9 thioester conjugate His6-tagged SUMO1(DG) was C-terminally functionalized with an alkyne group by an aminolysis reaction of an intein-generated SUMO thioester with propargylamine (Pa; Scheme 2). This SUMO construct lacks the terminal glycine residue (replaced by Pa in the modified version). To install the azide, p-azidophenylalanine (AzF) was incorporated in place of the active-site cysteine (C93) of Ubc9 by the nonsense-suppression method.[14] For purification purposes, Ubc9 was N-terminally fused to a streptavidin-binding peptide (SBP) tag. The presence and reactivity of the alkyne and azide functional groups were confirmed by CuAAC reactions with the fluorophores fluoresceinazide (Fl-N3) and dansylamide-alkyne (Figure 1 A, lanes 2 and 3). Incubation of His6-SUMO1(DG)-Pa and SBP-Ubc9(C93AzF) in a 1:2 molar ratio for 30 min under CuAAC conditions then led to the formation of the triazole93-linked protein conjugate (Figure 1 A, lane 4). Densitometric analysis of the gel band intensities revealed an efficiency of about 55 % for this reaction (calculated relative to the amount of the used SUMO1-alkyne reaction partner). After subsequent conjugate purification by affinity chromatography on Ni-NTA and streptactin sepharose, the 2
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Scheme 2. Preparation of a stable Ubc9–SUMO1 conjugate by click chemistry. SUMO1 lacking the last glycine residue (SUMO1(DG)) was prepared as a SUMO1–intein thioester and used for C-terminal modification with propargylamine (Pa). 2-Mercaptoethanesulfonate sodium was added as a thiol catalyst to enable formation of the SUMO–Mesna thioester. Catalytic cysteine 93 of Ubc9 was replaced by p-azidophenylalanine (AzF). Following the CuAAC reaction, the conjugate was purified, and the N-terminal streptavidinbinding peptide (SBP) tag of Ubc9 was removed by TEV protease. The latter step exposed an almost native N terminus of Ubc9 (Met1 replaced with Gly as the only sequence alteration). a) CuSO4, TBTA, TCEP; b) Ni-NTA and streptactin purification; c) TEV digest.
N-terminal SBP-tag of Ubc9 was proteolytically removed by TEV protease digest (Scheme 2 and Figure 1 B).
The triazole93-linked SUMO1(DG)–Ubc9 conjugate specifically binds to the RanBP2–E3 ligase complex
The triazole93-linked SUMO1–Ubc9 conjugate is stable under reducing conditions, and to E3 ligase and isopeptidase activities
Next, we investigated whether the triazole93-linked conjugate represents a suitable surrogate for the native SUMO1–Ubc9 thioester. To this end, interaction studies with the multi-subunit RanBP2–E3 ligase complex[13] were performed. To investigate the binding of the triazole93-linked SUMO1(DG)-Ubc9 conjugate to the RanBP2 complex, conjugate and reconstituted E3 ligase were preincubated on ice overnight then samples were analyzed by size-exclusion chromatography. The triazole conjugate co-eluted with the RanBP2 complex from the column, thus indicating efficient binding to E3 ligase (Figure 3 B, lane 8). Free wild-type (wt) Ubc9,free wt
With the triazole93-linked SUMO1(DG)–Ubc9 conjugate in hand, we wanted to test its behavior under conditions where working with the endogenous thioester is difficult. First, the stability of the conjugate under reducing conditions was tested by incubation in DTT-containing buffer. As a control, a Ubc9 ~ SUMO1 thioester was preformed in vitro. The latter displayed quantitative decomposition, but the triazole conjugate was completely stable in the presence of DTT (Figure 2 A). ChemBioChem 0000, 00, 0 – 0
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Next, we assessed the stability of the triazole conjugate in the presence of a SUMO E3 ligase: the multi-subunit RanBP2–E3 ligase complex.[13] This complex is composed of SUMO1-modified RanGAP1 and Ubc9 that bind synergistically to the RanBP2–E3 ligase region between Ran-binding domains RB3 and RB4 (Figure 3 A). The E3 ligase region itself consists of two 50-amino-acid internal repeats (IR1 and IR2) connected by a short linker (M). Formation of the RanBP2/RanGAP1*SUMO1/Ubc9 complex has been shown to take place at IR1, supported by a SUMO-interacting motif (SIM) at the N terminus of IR1.[13, 15] Of note, the Ubc9 molecule in this complex has only a structural (noncatalytic) function; binding of a second SUMO1-thioester-charged Ubc9 molecule within the IR2 region is required for catalysis[13] (Figure 3 A). As expected, the preformed Ubc9 ~ SUMO1 thioester quickly discharged when incubated with the RanBP2–E3 ligase complex; this gave rise to multi- and or poly-SUMOylated species of the RanBP2 protein. In contrast, the triazole conjugate displayed stability after incubation with the RanBP2 complex (Figure 2 B). Finally, we wanted to test the triazole93-linked SUMO1(DG)–Ubc9 conjugate and an isopeptide-conjugated thioester mimic for suitability under experimental conditions where SUMO isopeptidases might be present. The latter conjugate was prepared in analogy to the recently reported Ub–E2 isopeptide linkage,[7] by mutating the catalytic cysteine of Ubc9 to lysine and the adjacent lysine (101) to arginine (to ensure uniform modification on a single lysine) and subsequent E1-dependent in vitro SUMOylation of the Ubc9(C93K/K101R) construct (data not shown). Both thioester surrogates were incubated with a catalytic fragment of the SUMO isopeptidase SENP1 (SENP1cat, sentrin-specific protease; Figure 2 C). The SUMO1–Ubc9(C93K/K101R) conjugate was slowly cleaved under the applied conditions, but the triazole-linked SUMO1(DG)-Ubc9 conjugate prepared by CuAAC was completely stable in the presence of SENP1cat (Figure 2 C).
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Full Papers azole93-linked SUMO1(DG)–Ubc9 conjugate to this RanBP2 complex mutant was significantly impaired (Figure 3 D, lane 7) compared to the unmutated complex (lane 5). Finally, we compared the binding of the triazole conjugate to RanBP2 complexes with that of the isopeptide-based thioester mimic. These mimics showed very similar behavior towards the E3 ligase complex, as they co-migrated to similar extent with the wt RanBP2 complex and showed similarly reduced interaction with the RanB(IR2mut) complex (Figure 3 D, lanes 6 and 8).
Conclusion We have demonstrated the suitability of CuAACmediated chemical conjugation to generate a chemically, proteolytically, and E3 ligase stable analogue of the highly reactive SUMO1 ~ Ubc9 thioester intermediate formed during enzymatic SUMO conjugation. Alkyne-functionalized SUMO1 and azide-modiFigure 1. Synthesis of a triazole93-linked SUMO1(DG)–Ubc9 thioester conjugate. fied Ubc9 were obtained by recombinant expression A) CuAAC reactions were performed for 30 min at RT and analyzed by SDS-PAGE followed and rapidly conjugated by the CuAAC reaction in by Coomassie staining (left) and UV illumination (right). (*: unidentified by-product.) good yields. The chemical conjugate His6-SUMO1-triB) TEV protease digest of purified His6-SUMO1(DG)-triazole93-SBP-Ubc9 was performed with GST-TEV for 64 h at 4 8C (protease/protein 1:5 (w/w); lanes 1 and 2). The resulting azole93-SBP-Ubc9 could be liberated from an SBP tag His6-SUMO1(DG)-triazole93-Ubc9 was then purified by Ni-NTA chromatography (lane 3: by cleavage with TEV protease and purified. Interacwashing fraction; lanes 4–7: elution fractions). See Scheme 2 for location of the TEV site tion studies with the SUMO-specific RanBP2–E3 ligase in the chemically conjugated protein. complex revealed co-elution of the triazole93-linked conjugate with the complex by size-exclusion chromatography. A single point mutation in the SIM binding groove of the chemically conjugated SUMO1 resulted in SUMO1 and free His6-SUMO1 (negative controls) showed no a significant reduction of this interaction, consistent with the stable interaction with the RanBP2 complex under these condiobservation that SUMOylation reactions on Borealin with the tions (Figure 3 C, lane 3 and 3 B, lanes 5 and 6, respecctively). corresponding SUMO1 mutant are much less efficient than To differentiate between free Ubc9 and the Ubc9 molecule in with wt SUMO1. Importantly, binding of the triazole93-linked the RanBP2 complex, we used a RanBP2 complex containing a C-terminally HA-tagged Ubc9-variant (Figure 3 C). Note that conjugate was also impaired when using a RanBP2 complex the presence of the HA-tag had no effect on the binding propvariant in which the IR2 sequence of RanBP2 was mutated to erties of the triazole conjugate (Figure 3 C, lane 3). eliminate the catalytic center of the E3 ligase. To address the specificity of the interaction with the RanBP2 Taken together, these data suggest that the triazole93-linked complex, we also prepared a triazole93-linked Ubc9 conjugate SUMO1–Ubc9 conjugate functions as a suitable structural mimic for the native SUMO1 ~ Ubc9 thioester. The observed containing a SUMO1(F36L) mutant. F36 participates in SUMO– binding of the chemical SUMO1–Ubc9 conjugate to the preSIM binding,[16] and it has been shown that transfer of this SUMO mutant to the endogenous target protein Borealin in assembled RanBP2/RanGAP1*SUMO1/Ubc9 complex supports RanBP2-complex-mediated SUMOylation reactions is signifiour previous finding that two Ubc9 molecules are required for cantly impaired compared to wt SUMO1.[13] Consistent with RanBP2-complex-dependent SUMOylation:[13] the Ubc9 in the this, reduced RanBP2 complex binding was observed for the complex serves a structural role, and the Ubc9 binding triazole93-linked SUMO1(F36L) (DG)–Ubc9 conjugate (Figure 3 B, a SUMO modifier in thioester linkage is the catalytically important E2 conjugating enzyme. The triazole linkage appears to lane 7). provide sufficiently correct positioning and orientation of To gain further evidence for a specific interaction of the SUMO1 on the Ubc9 scaffold, although it is slightly longer chemical thioester analogue with the E3 ligase complex we than the native thioester linkage (Scheme 1 B and D) and conincluded the RanBP2 (IR2mut) complex variant in our binding tains a different pattern of freely rotatable and conformationalassays (Figure 3 D). This variant contains a RanBP2 fragment ly restricted bonds. In line with this interpretation, binding of with five mutations in the IR2 region (implicated in catalysis in the triazole mimic to the RanBP2 complex was very similar to the assembled E3 ligase complex): a double mutation (I2711A/ that of the isopeptide-based thioester mimic (Figure 3 D). Thus, I2712A) in the putative SIM2 and a triple mutation (L2729A/ chemical conjugation to stably attach SUMO to target proteins L12731A/F2736A) at residues crucial for binding of Ubc9 and by the CuAAC reaction was expanded from mimics of isopepthus for catalytic activity.[13, 15] As expected, binding of the tri-
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Full Papers ampicillin (100 mg mL 1), kanamycin (50 mg mL 1), and chloramphenicol (34 mg mL 1). Synthetic oligonucleotides were purchased from Biolegio (Nijmegen, The Netherlands). Site-directed mutagenesis was performed according to the QuikChange Lightning Site-Directed Mutagenesis protocol (Agilent Technologies). All created plasmids were verified by DNA sequencing (GATC Biotech, Konstanz, Germany, and Seqlab, Gçttingen, Germany). The anti-Ubc9 antibody (sc-10759) was from Santa Cruz Biotechnology (Heidelberg, Germany). For anti-RanBP2, see ref. [18]. Expression plasmids: Plasmid pSS19 (encoding His6-SUMO1(DG)GyrA-CBD) was created from pNW12 (encoding His6-SUFigure 2. Stability of the triazole93-linked SUMO1–Ubc9 conjugate. A) Comparison of the stability of the triazole93MO1(DGG)-Gyr-CBD)[8] by site-dilinked His6-SUMO1(DG)–Ubc9 conjugate and a preformed SUMO1–Ubc9 thioester in the presence of DTT. rected mutagenesis to insert one SUMO1–Ubc9 thioester was preformed with 1.6 mm SUMO E1 enzyme, 8 mm Ubc9, and 24 mm SUMO1 at 30 8C for carboxy-terminal glycine residue 10 min. The preforming reaction was quenched by addition of 60 mm EDTA. Thioester formation showed a yield into SUMO1(DGG) by using oligoof about 20 % (first lane). According to this estimation, 300 nm thioester and His6-SUMO1(DG)-triazole93-Ubc9 nucleotides 5’-GTTTAT CAGGAA were analyzed with non-reducing (50 mm Tris (pH 6.8), 4 m urea, 10 % (v/v) glycerol, and 2 % (w/v) SDS; “ DTT”) GGCTGC ATCACG CAAACG and reducing sample buffer (50 mm Tris (pH 6.8), 2 % (w/v) SDS, 0.1 % (w/v) Bromophenol Blue, 10 % (v/v) glycerol, GGAGAC-3’ (forward, inserted Gly and 100 mm DTT; “+DTT”) by anti-Ubc9 immunoblotting. B) Comparison of the stability of the triazole93-linked codon underlined) and 5’-GTCTCC SUMO1(DG)–Ubc9 conjugate and a preformed SUMO1–Ubc9 thioester towards the RanBP2–E3 ligase complex. SUMO1–Ubc9 thioester was preformed as in A); 300 nm thioester and His6-SUMO1(DG)-triazole93-Ubc9 were incuCGTGAT GCAGCC CGTTTG TTCCTG bated with 50 nm reconstitued RanBP2 complex at 30 8C for 15 min. Samples were analyzed in non-reducing ATAAAC-3’ (reverse). pSS19 was sample buffer by anti-Ubc9 and anti-RanBP2 immunoblotting. (#: excess free Ubc9 from the preforming reaction.) then used a template for a site-diC) Comparison of the stability of the triazole93-linked SUMO1(DG)-Ubc9 conjugate and an isopeptide-linked rected mutagenesis to create the SUMO1-Ubc9(C93K/K101R) conjugate towards SUMO isopeptidase. His6-SUMO1(DG)-triazole93-Ubc9 was incubated F36L mutation into the with various concentrations of SENP1cat (aa 419–644) at 30 8C for 30 min. The isopeptide-linked SUMO1– SUMO1(DG) gene by using oligoUbc9(C93K/K101R) conjugate was treated the same way. Reactions were analyzed by anti-Ubc9 immunoblotting. nucleotides 5’-GATAGC AGTGAG ATACAT CTGAAA GTGAAA ATGACA AC-3’ (forward, L36 codon underlined) and 5’-GTTGTC ATTTTC tide bonds[8, 10] to a mimic for the active-site thioester of an E2 ACTTTC AGATGT ATCTCA CTGCTA TC-3’ (reverse) to create pSS38 enzyme. (His 6-SUMO1(F36L)(DG)-GyrA-CBD). To generate pSS34 (encoding In general, non-hydrolysable Ub/Ubl–E2 thioester mimics pSBP-TEVsite-Ubc9(C93TAG)), a TAG codon was inserted at position prepared by CuAAC provide exciting prospects as novel tools C93 into the Ubc9 open reading frame by site-directed mutageneto enable biochemical and structural characterization of prosis with oligonucleotides 5’-CTTCTG GCACAG TGTAGC TGTCCA tein complexes involving E3 ligases, and potentially even subTTCTAG AGGAAG AC-3’ (forward, TAG codon underlined) and 5’strate proteins. This approach is especially attractive in cases GTCTTC CTCTAG AATGGA CAGCTA CACTG-TGCCAG-AAG-3’ (rewhere conventional strategies to obtain stable thioester verse). From the resulting vector pSS13 (pUbc9C93TAG) the Ubc9C93TAG gene was then amplified by using oligonucleotides mimics are not applicable (e.g., the SUMO E2 enzyme Ubc9, 5’-CCGCTC GAGGAA AACCTG TATTTT CAGGGC TCGGGG ATCGCC for which an oxyester is unstable). The resistance of the triaCTCAGC-3’ (TEV cleavage site underlined, XhoI restriction site in zole linkage (in contrast to the isopeptide linkage) towards isoitalics) and 5’-CCCAAG CTTTTA TGAGGG GGCAAA C-3’ (HindIII repeptidase activity will also enable the use of these mimics for striction site in italics). The SBP gene was amplified from pSS02 interaction studies in cell lysate without the need to inactivate (pSBP-HA-GpD-PML11(TAG))[10a] by using oligonucleotides 5’-CATGCC isopeptidases by harsh treatments, such as exposure to high ATGGAC GAAAAA ACCACC-3’ (forward, NcoI site in italics) and 5’concentration of N-ethylmaleimide (NEM). Such experiments CCGCTC GAGGCC GGGTTC ACGCTG AC-3’ (reverse, XhoI site in italmight help to identify previously unknown E3 ligases or other ics). The PCR products were digested with the appropriate enzymes and ligated simultaneously into an NcoI/HindIII digested cellular components interacting with Ub- or Ubl-loaded E2 enpET28a-vector to give pSS34. Plasmids for RanBP2RB3 4 (aa 2304– zymes. 3062), hRanGAP1, wt SUMO1, wt Ubc9, and GST-SENP1cat (aa 419– 644) have been described previously.[13, 17]
Experimental Section
Protein production and purification: For the expression of His6SUMO1(DG)-GyrA-CBD and His6-SUMO1(F36L)(DG)-GyrA-CBD, Escherichia coli BL21(DE3) cells were transformed with the respective
General: Standard molecular biology protocols were applied for DNA cloning and expression. The antibiotics were
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Full Papers EDTA (1 mm)). After washing with buffer W, the protein was eluted with buffer E (Tris (100 mm, pH 8.0), NaCl (150 mm), EDTA (1 mm), desthiobiotin (2 mm)) followed by dialysis in dialysis buffer (HEPES (20 mm, pH 8.0), NaCl (150 mm)). In vitro reconstitution of the RanBP2 complex (His6-RanBP2RB3 4 fragment (aa 2304–3062), fulllength untagged RanGAP1*SUMO1, and full-length Ubc9) was performed as previously reported.[13] CuAAC and purification of conjuSUMO1-triazole93-Ubc9 gates: Reactions were performed in dialysis buffer. SBP-TEVsiteUbc9(C93AzF) (20 mm) and Pamodified SUMO1(DG) (10 mm) were mixed and treated with Tris(2-carboxyethyl)phosphine (TCEP; 500 mm). Then Tris(benzyltriazolyl-methyl)amine (TBTA, 50 mm; in DMSO/tBuOH (1:4)) and CuSO4 (500 mm) were added. After incubation for 30 min at room temperature, the reaction was stopped by addition of EDTA (10 mm). Control reactions with Figure 3. The triazole93-linked SUMO1-Ubc9 thioester analogue specifically binds to the RanBP2–SUMO E3 ligase the fluorophores N-(3-azidopropcomplex. A) Schematic representation of the multi-subunit RanBP2/RanGAP1*SUMO1/Ubc9 E3 ligase complex. The yl)-fluorescein-amide (CLK-FA005-1, complex was in vitro reconstituted from the indicated RanBP2RB3 4 fragment (aa 2304–3062: Ran-binding sites Jena Bioscience, Jena, Germany) RB3 and RB4 and the E3 ligase domain (IR1-M-IR2)). A putative SIM (SIM2) is at the N terminus of IR2. B) Gel filtration interactions studies. The RanBP2 complex was preincubated with triazole93-linked conjugates and the indicatand dansylamide-alkyne[8] (both ed SUMO1 variants (2 mm) on ice overnight, then samples were run on a Superdex 200 column. Fractions at 0.42 dissolved in DMF) were performed column volume were analyzed with Coomassie-stained gels. C) Binding study as above for a RanBP2 complex similarly, but in the dark. All reacwith C-terminally HA-tagged Ubc9, the triazole93-linked conjugate, and Ubc9. D) Binding study as above for the tions were analyzed by SDS-PAGE RanBP2 (IR2mut) complex variant and the isopeptide-linked SUMO1–Ubc9(C93K/K101R) conjugate, as well as the with UV illumination. The efficiency triazole93-linked conjugate. (RanBP2 (IR2mut) = I2711A/I2712A/L12729A/L12731A/F2736A; *: ovalbumin in the of the CuAAC reaction was deterbuffer.) mined by densitometric analysis of band intensities of a Coomassiestained SDS-PAGE gel with Scion Image (Scion Corporation) plasmids and cultured in LB medium supplemented with ampicillin software. Percentage of triazole conjugate formation was calculatat 37 8C to OD600 = 0.6, then protein expression was induced with ed relative to the amount of the used SUMO1-alkyne reaction partIPTG (0.4 mm) for 4 h at 28 8C. To incorporate p-azidophenylalanine ner. (AzF), E. coli BL21 (DE3) cells were co-transformed with the plasmid encoding SBP-TEVsite-Ubc9(C93TAG) and the plasmid providing the To purify His6-SUMO1(DG)-triazole93-SBP-TEVsite-Ubc9 conjugates, genetic information for the orthogonal AzFRS/AzFtRNACUA pair deCuAAC reaction mixtures were dialyzed against buffer W supplerived from Methanococcus jannashii (kindly provided by Peter G. mented with DTT (2 mm). After purification over streptactin seSchultz, Scripps Institute, La Jolla, CA). Cells were grown in M9 minpharose (as above), pooled elution fractions were dialyzed against imal medium (Na2HPO4 (45 mm), KH2PO4 (25 mm), NaCl (8.5 mm), Ni-NTA buffer A (Tris (50 mm, pH 8.0), NaCl (300 mm)) with b-merMgSO4 (1 mm), CaCl2 (0.1 mm), FeCl3 (22 nm), thiamine captoethanol (2 mm) and immobilized on Ni-NTA sepharose 1 (0.03 mg mL ), NH4Cl (0.1 %), glucose (0.2 %)) supplemented with (5Prime, Hamburg, Germany). After washing with buffer A supplekanamycin and chloramphenicol to OD600 = 0.6. AzF (1 mm; mented with imidazole (20 mm then 40 mm), elution was perBachem, Weil am Rhein, Germany) was added, and protein expresformed by addition of buffer A supplemented with imidazole sion was induced with IPTG (1 mm) for 4 h at 37 8C. (250 mm). To cleave the N-terminal SBP-sequence of Ubc9, purified Cells were lysed by two passages through a C5 emulsifier (Avestin, conjugates were mixed with GST-TEV (protein/protease 5:1, w/w) Ottawa, ON). Purification of CBD-tagged SUMO proteins and subseand dialyzed against TEV cleavage buffer (Tris (50 mm, pH 8.0), quent C-terminal modification with propargylamine (Pa) were perEDTA (0.5 mm), DTT (1 mm)) at 4 8C for 64 h to allow complete formed as previously described.[8, 10a] SBP-TEVsite-Ubc9(C93AzF) was conversion. To remove free SBP and the GST-TEV, the His6immobilized on streptactin sepharose (IBA, Gçttingen, Germany) SUMO1(DG)-triazole93-Ubc9 conjugate was immobilized on Ni-NTA sepharose and finally dialyzed against assay buffer (HEPES-KOH equilibrated in buffer W (Tris (100 mm, pH 8.0), NaCl (150 mm),
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Preparation of isopeptide93-linked SUMO1-Ubc9-conjugate: For formation of the isopeptide-linked conjugate, C93 (catalytic cysteine of Ubc9) was mutated to lysine. In order to achieve specific modification at residue 93, Lys101 was additionally mutated to arginine. The isopeptide-linked conjugate was formed by a SUMOylation reaction with a mixture of Ubc9 (C93K/K101R) (50 mm), SUMO1 (50 mm), SUMO E1 enzyme (500 nm; both prepared in the lab), and ATP (5 mm) in mimic buffer (Tris-HCl (50 mm, pH 10.0), NaCl (150 mm), MgCl2 (5 mm), DTT (1 mm), aprotinin, leupeptin, pepstatin (1 mg mL 1 each; Biolmol)). The reaction was incubated for 18–24 h at 30 8C. The mimic was subsequently purified by size exclusion chromatography on a Superdex75 GL gel filtration column equilibrated in assay buffer supplemented with aprotinin, leupeptin, pepstatin (1 mg mL 1 each). In vitro formation of a SUMO1–Ubc9 thioester: SUMO1–Ubc9 thioester was formed with SUMO E1 enzyme (1.6 mm), Ubc9 (8 mm), and SUMO1 (24 mm) in assay buffer (but with 0.1 mm DTT not 1 mm) supplemented with ovalbumin (0.2 mg mL 1), Tween 20 (0.05 %, v/v), and aprotinin, leupeptin, pepstatin (1 mg mL 1 each) at 30 8C for 10 min. The reaction was quenched by addition of EDTA (60 mm). E3 ligase stability assay: Triazole93-linked SUMO1(DG)-Ubc9 (300 nm) and preformed SUMO1–Ubc9 thioester (300 nm) were incubated with RanBP2 complex (50 nm) in the above buffer for 15 min at 30 8C. Samples were analyzed by immunoblotting with anti-Ubc9 and anti-RanBP2 antibodies. SENP1 assay: Triazole93-linked SUMO1(DG)-Ubc9 (1 mm) and SUMO1-Ubc9(C93K/K101R) (1 mm) were incubated in assay buffer supplemented with ovalbumin (0.2 mg mL 1), Tween 20 (0.05 %, v/ v), and aprotinin, leupeptin, pepstatin (1 mg mL 1 each) for 30 min at 30 8C with different concentrations of GST-SENP1cat (0, 2, 20, or 200 nm). Samples were analyzed by anti-Ubc9 immunoblotting. Gel filtration binding assays with the RanBP2–E3 ligase complex: RanBP2 complexes were incubated with triazole93-linked SUMO1(DG)-Ubc9 conjugate (2 mm), Ubc9 wt (2 mm), and SUMO1 wt (2 mm) in assay buffer supplemented with ovalbumin (0.2 mg mL 1), Tween 20 (0.05 %, v/v), and aprotinin, leupeptin, pepstatin (1 mg mL 1 each) on ice overnight. Samples were applied on a Superdex 200 5/150 GL gel filtration column equilibrated in assay buffer supplemented with aprotinin, leupeptin, pepstatin (1 mg mL 1 each). Fractions at 0.42 column volume were analyzed by Coomassie-stained gels.
Acknowledgements We thank Nadine D. van Treel for discussions and Peter G. Schultz (The Scripps Research Institute) for providing plasmids. H.D.M. and F.M. acknowledge funding by the Deutsche Forschungsgemeinschaft (SFB858, GRK1188 and SFB638, respectively). Keywords: biological activity · click chemistry · enzyme catalysis · post-translational modifications · SUMO · ubiquitin [1] a) A. Hershko, A. Ciechanover, Annu. Rev. Biochem. 1998, 67, 425 – 479; b) O. Kerscher, R. Felberbaum, M. Hochstrasser, Annu. Rev. Biochem. 2006, 22, 159 – 180; c) A. Flotho, F. Melchior, Annu. Rev. Biochem. 2013, 82, 357 – 385.
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FULL PAPERS S. Sommer, T. Ritterhoff, F. Melchior,* H. D. Mootz*
Chemical camouflage: Ubiquitin and SUMO bound as thioesters to their E2 conjugating enzymes are labile intermediates in the presence of an E3 ligase, a fact that hampers biochemical characterization of the charged E2/E3 complexes. A stable chemical conjugate of SUMO1 with the SUMO E2 enzyme is now reported as a thioester mimic to address these limitations.
&& – && A Stable Chemical SUMO1–Ubc9 Conjugate Specifically Binds as a Thioester Mimic to the RanBP2–E3 Ligase Complex
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