Special Issue Article Received: 13 September 2013
Revised: 23 October 2013
Accepted: 24 October 2013
Published online in Wiley Online Library: 15 December 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2591
SUMOylated RanGAP1 prepared by click chemistry‡ Nadine D. van Treel and Henning D. Mootz* Ubiquitin and ubiquitin-like proteins such as SUMO represent important and abundant post-translational modifications involved in many cellular processes. These modifiers are reversibly attached via an isopeptide bond to lysine side chains of their target proteins by the action of specific E1, E2, and E3 enzymes. A significant challenge in studying ubiquitylation and SUMOylation is the frequently encountered inability to access desired conjugates at a defined position of the target protein and in homogenous form by using enzymatic preparation. In recent years, several chemical conjugation approaches have been developed to overcome this limitation. In this study, we aimed to selectively SUMOylate a 189-amino acid fragment of human RanGAP1 (amino acids 398–587) at the position of Lys524 by applying two recently reported approaches based on the Cu(I)-catalyzed alkyne-azide cycloaddition. Because of low yields observed for the incorporation of an unnatural amino acid with an azide moiety by the tRNA suppression technology, this route was abandoned. However, installing a single cysteine at position 524 and its selective alkylation was successful to introduce the azide group. The triazole-linked SUMO1**RanGAP1 conjugate could be obtained in good yields, purified, and was shown to specifically interact with RanBP2/Ubc9. Thus, we expand the scope of proteins accessible to chemical conjugation with ubiquitin-like proteins and underline the importance of having alternative approaches to do so. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: bioorthogonal; click chemistry; CuAAC; post-translational modification; protein chemistry; SUMO; ubiquitin
Introduction
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* Correspondence to: Henning D. Mootz, Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany. E-mail: henning. [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3–6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany Abbreviations: aa, amino acids; AzF, p-azidophenylalanine; CuAAC, coppercatalyzed alkyne-azide cycloaddition; NTA, nitrilotriacetic acid; RF1, release-factor 1; SUMO, small ubiquitin-like modifier; TBTA, tris(benzyltriazolylmethyl)amine; TCEP, tris(2-carboxyethyl)phosphine; Ub, ubiquitin; Ubl, ubiquitin-like protein; LB, lysogeny broth; IPTG, isopropyl beta-D-1-thiogalactopyranoside; TB, transport buffer; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GST, glutathion S-transferase; PML, promyelocytic leukemia protein.
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The post-translational modification of proteins with ubiquitin (Ub) and Ub-like proteins (Ubls) such as small Ub-like modifier (SUMO) plays an important role in regulation of numerous processes in higher organisms, for example, proteasomal degradation, cell-cycle control, and regulation of signaling processes [1–3]. Ubls are typically attached to a specific lysine side chain of the target or substrate protein by an isopeptide bond involving their C-terminal carboxylate group. Ub and SUMO can also form chains by attachment of an additional post-translational modifier onto one of the seven lysines or the N-terminal amino group within Ub itself [4], or within Lys11 in the case of the human SUMO2/3 isoforms [5–7], respectively. The formation of Ubl conjugates is catalyzed by the action of dedicated E1, E2, and E3 enzymes and counteracted by specific isopeptidases, such as the deubiquitylases and the SUMO-deconjugating enzymes (e.g. the sentrin-specific protease proteins in humans), thereby rendering this process reversible [2,3,8]. Importantly, because the pattern of post-translational modification relies on the substrate specificity, activity, and localization of the conjugating enzymes and is not directly genetically encoded, it is impossible to predict Ubl conjugates with certainty or to obtain them by simple recombinant protein production. In fact, most conjugates cannot be prepared by enzymatic means at all, because not all E2 and E3 enzymes are sufficiently characterized and because enzymatic preparation can lead to mixtures and incomplete modifications at desired positions. However, in order to understand the biochemical, structural, and cellular consequences of Ubl modification, protein preparations with a defined attachment of the modifier at a desired position and in homogeneous form are required. For these reasons, significant research efforts have been
undertaken by several groups in recent years to develop nonenzymatic routes to ubiquitylated or SUMOylated proteins. Several chemical conjugation strategies have been reported that enable the preparation of modified proteins in homogeneous form with freely chosen attachment points [9–11]. Bond formation between the protein of interest and Ub/SUMO was achieved by alkylation [12], native and isopeptide chemical ligation [13–18], amide coupling [19], disulfide formation [20,21], thiol-ene addition [22], oxime ligation [23], and copper-catalyzed alkyne-azide cycloaddition (CuAAC, or click reaction) [24–27]. These approaches can be categorized into those yielding the native structure of the isopeptide bond and those giving rise to analogous linker structures. Furthermore, in some cases, the protein of interest is accessed by total chemical synthesis, whereas in others, it is obtained by recombinant protein expression.
VAN TREEL AND MOOTZ
Scheme 1. Two approaches for synthetic Ubl conjugates prepared by click chemistry. Ubl proteins can be ubiquitin and SUMO, for example. (A) Overview of the two approaches to incorporate the azido moiety into the recombinant substrate protein. CuAAC reaction with the C-terminally alkyne-modified Ubl modifier gives the desired conjugate. Note that either one or two of the conserved C-terminal glycines on the Ubl were deleted for approaches 2 and 1, respectively, to best resemble the length of the native isopeptide bond. (B) Preparation of the alkyne-modified Ubl by using an intein fusion protein to generate the reactive thioester (Pa = propargylamine).
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We [24–26] and others [27] have previously explored the CuAAC reaction [28,29] to attach the Ubl by a triazole linkage to the protein of interest. To this end, two approaches were developed by us (Scheme 1). In both cases, the C terminus of the Ubl was functionalized with an alkyne moiety by aminolysis of an intein thioester with propargylamine (Scheme 1B), but two different routes for incorporating the azide group were explored. In the first approach, the acceptor lysine in the substrate protein was mutated to a cysteine, and the thiol moiety of this amino acid was subsequently used to introduce the azide group for the click reaction [24]. In order to achieve regioselectivity, the introduced cysteine has to be the only cysteine in the substrate protein, or at least the only cysteine accessible and reactive under the conditions used. To circumvent this limitation, we developed the second approach (Scheme 1A) in which the azide group is introduced by unnatural amino acid mutagenesis [30,31] using the amber suppression technology [25,26]. Specifically, p-azidophenylalanine (AzF) was introduced at the desired position and could be used in the click reaction even in the presence of other cysteines in the same protein, as shown for the synthesis of a SUMO conjugate with the E2-conjugating enzyme Ubc9 [25]. For both approaches, we could show that the copper catalyst was not harmful as long as its exposure to the proteins was restricted to 30–60 min. Thus, the conjugation reactions could be performed under non-denaturing conditions. This point is important, because a key advantage of both approaches is that the substrate protein can be prepared recombinantly and thus does
not underlie any size restrictions that have to be considered in alternative fully synthetic approaches. Furthermore, the stability of both triazole linkers against hydrolytic cleavage by isopeptidases [24,26] holds a great potential for pulldown studies in cell extracts, for example. Our biochemical data reported so far on a PML-SUMO2, a Ubc9-SUMO2, and seven Ub–Ub conjugates and their interaction partners support the idea that both triazole linkers are good PML-SUMO2 [24], a Ubc9-SUMO2 [25], and seven Ub-Ub conjugates [26] and their structural mimics of the native isopeptide bond. This view could be further strengthened by QM/MM calculations for the triazole linker involving the unnatural amino acid AzF [32]. Ran GTPase activating protein (RanGAP1) was the first SUMOylated protein to be identified [33,34]. SUMO1 is one of the three human SUMO isoforms known to be conjugated to proteins. In contrast to the highly homologous SUMO2/3, SUMO1 does not form chains. SUMO1*RanGAP1 has been shown to be part of a stable pore complex with the Ran-binding protein RanBP2 at the nuclear envelope [33]. This interaction is enhanced by the presence of the E2-conjugating enzyme Ubc9, which exerts a synergistic effect on the complex formation [35,36]. It was shown that this complex forms an active SUMO E3 ligase [37]. The complex also plays an important role in transport processes across the nuclear membrane and in the cell cycle. Unmodified RanGAP1 and free SUMO1 molecules cannot interact with RanBP2 and fail to form the complex [33]. In this work, we aimed to further show the general applicability of the CuAAC conjugation approaches to other substrate proteins
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CLICK SUMOylation and synthesized a RanGAP1-SUMO1 conjugate. We found that the amber suppression technology to insert the unnatural amino acid AzF performed only very poorly for the RanGAP1 fragment of 189 amino acids (aa) in size. However, introducing the azide at the desired position via a cysteine was successful and underlines the versatility of the two CuAAC approaches for Ubl conjugation.
Materials and Methods General Standard molecular biology procedures were applied for DNA cloning and expression. Antibiotics were used at 100 μg/ml (ampicillin) and 30 μg/ml (chloramphenicol). Synthetic DNA oligonucleotides were purchased from Biolegio (Nijmegen, the Netherlands). All DNA vectors were confirmed by DNA sequencing (GATC Biotech, Konstanz, Germany). Site-directed mutagenesis was performed according to the QuickChange protocol from Stratagene. Cloning of Expression Plasmids pNW12 for the expression of His6-SUMO1(ΔGG)-GyrA-CBD was cloned as previously described [24]. pNW26 for the expression of His6-RanGAPC573S: Using site-directed mutagenesis, a serine codon was introduced at the position of the C573 codon with oligonucleotides 5′-CCCTGGAATCCTCGTCCTTCGCCCGC-3′ (forward; serine codon underlined) and 5′-GCGGGCGAAGGACGAGGATTCCAGGG-3′ (reverse) in the plasmid encoding for human RanGAP1 (aa 398– 587) [38]. pNW27 for expression of His6-RanGAPC573S,K524C: pNW26 served as a template for the site-directed mutagenesis to change the K524 codon to a cysteine codon using primers 5′CACATGGGTCTGCTCTGCAGTGAAGACAAGGTC-3′ (forward; cysteine codon underlined) and 5′-GACCTTGTCTTCACTGCAGAGCAGACCCATGTG-3′ (reverse). pNW35 for expression of His6-RanGAPK524AzF: An amber stop codon was inserted at the position of K524 in the abovementioned plasmid encoding human RanGAP1 using primers 5′-CACATGGGTCTGCTCTAGAGTGAAGACAAGGTC-3′ (forward; stop codon underlined) and 5′- GACCTTGTCTTCACTCTAGAGCAGACCCATGTG-3′ (reverse). Protein Expression and Purification
Cysteine Alkylation Construct 3 (His6-RanGAP1C573S,K524C; ~50 μM, pH 7.0) was treated for 20 min with 5 eq. tris(2-carboxyethyl)phosphine (TCEP), followed by addition of 20 eq. iodoacetamide ethyl azide [24]. After 10 min, again 5 eq. TCEP were added, and steps repeated twice. After 3.5 h total reaction time, 60 eq. DTT were added to quench excess iodoacetamide, and the protein was dialyzed against dialysis buffer (20 mM HEPES, 150 mM NaCl, pH 8.0).
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Protein expression was performed in Escherichia coli BL21(DE3) cells, which were transformed with the corresponding plasmids. All cells were grown in LB media supplemented with ampicillin at 37 °C to an OD600 of 0.6, and expression was induced with 0.4 mM IPTG and carried out for 4–5 h at 28 °C, unless otherwise noted. Expressions of GST-RanBP2 (GST explained in abbreviations) (aa 2553–2838, expression at 30 °C), Ubc9 (expression at 37 °C), and His6-RanGAP (aa 398–587) were performed using plasmids kindly provided by Frauke Melchior, ZMBH Heidelberg, Germany [38]. Coexpression of RanGAP1 proteins with SUMO1 was carried out in E. coli BL21(DE3) cells, which were co-transformed with the plasmids encoding for E1 and E2 enzymes as well as the native human His6-SUMO1 isoform (kindly provided by Gerrit Praefcke, University of Cologne, Germany) [39]. For expression of proteins with AzF [30], E. coli BL21(DE3) cells were co-transformed with the plasmid encoding the target gene containing the amber stop codon and the vector encoding the orthogonal pAzFRS/tRNACUA pair (kindly provided by Peter G. Schultz, Scripps Research Institute, La Jolla, USA). Cells were grown in M9-minimal media (45 mM Na2HPO4, 25 mM KH2PO4,
8.5 mM NaCl, 1 mM MgSO4, 0.1 mM CaCl2, 22 nM FeCl3, 0.03 mg/ml thiamine, 0.1% NH4Cl, 0.2% glucose) supplemented with 30 μg/ml chloramphenicol and 100 μg/ml ampicillin at 37 °C. When an OD600 of 0.6 was reached, AzF (purchased from Bachem, Bubendorf, Switzerland) was added to a final concentration of 1 mM, and protein expression was induced with 0.4 mM IPTG. Cultures were then incubated for another 4 h at 37 °C. All cells were harvested by centrifugation, resuspended in the respective buffer, and lysed by two passages through an EmulsiFlex-C5 (Avestin, Mannheim, Germany) emulsifier. In the case of Ubc9 expression, cells were harvested by centrifugation, resuspended in Ubc9 lysis buffer (50 mM Na2PO4, 50 mM NaCl, pH 6.5), and frozen at 80 °C. Thawing the cell pellet causes the protein to leak out of the cells, thus making cell lysis unnecessary and allowing for direct purification after removing cell debris by centrifugation [38]. Purification of chitin binding domain-tagged SUMO1 was carried out as previously described [24]. RanGAP1 proteins were purified on Ni-NTA agarose (Invitrogen, Darmstadt, Germany) equilibrated in Ni-NTA buffer A (50 mM Tris, 300 mM NaCl, pH 8.0) supplemented with 10 mM imidazole. After washing with Ni-NTA buffer A supplemented with 20 mM imidazole, a gradient was run ranging from 20 to 500 mM imidazole using an ÄKTA purifier (GE Healthcare, Munich, Germany). Cell lysate with GST-RanBP2 (aa 2553–2838) was incubated for 1 h at 4 °C with GST-bind resin equilibrated in RanBP2 lysis buffer [50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA)], washed extensively with RanBP2 wash buffer [50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT)], and then eluted using 20 mM glutathione in RanBP2 wash buffer. Fractions containing GST-RanBP2 were pooled and further purified by gel filtration using a Superdex 200 column (GE Healthcare, Munich, Germany) equilibrated in TB buffer [20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)2, 1 mM EGTA, 1 mM DTT, pH 7.3]. The purification of untagged Ubc9 [38] was performed via cation exchange chromatography at 4 °C using SP-sepharose (Fast Flow, Sigma-Aldrich, Schnelldorf, Germany) equilibrated in Ubc9 lysis buffer. After extensive washing with Ubc9 wash buffer (50 mM Na2PO4, 50 mM NaCl, 1 mM DTT, pH 6.5), the protein was eluted with Ubc9 elution buffer (50 mM Na2PO4, 300 mM NaCl, 1 mM DTT, pH 6.5). Fractions containing purified Ubc9 were pooled and dialyzed against TB buffer. All SUMO1-RanGAP1 conjugates were purified on Ni-NTA agarose as described earlier. After dialysis of the conjugates against anion exchange buffer A (20 mM Tris, pH 8.0), the conjugates were further purified by anion exchange chromatography by running a gradient from 0% to 100% buffer B (20 mM Tris, 500 mM NaCl, pH 8.0) in 30 min at a flow rate of 1 ml/min on a 1 ml MonoQ HP column (GE Healthcare, Munich, Germany). Proteins were then dialyzed in pulldown buffer [20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)2, 1 mM EGTA and 0,05% Tween-20, pH 7.3] and stored at 80 °C. All protein concentrations were determined using the calculated absorbances at 280 nm.
VAN TREEL AND MOOTZ Cu(I) Catalyzed Azide-Alkyne Cycloaddition (CuAAC) [28,29] The fluorophores tetramethylrhodamine-azide and dansylamidealkyne (in DMF) were mixed in equimolar amounts (20 μM each) with corresponding reaction partners and treated with 500 μM TCEP. Then, tris(benzyltriazolylmethyl)amine (TBTA) at 50 μM and CuSO4 at 500 μM final concentrations were added to the solution. The CuAAC reaction was carried out for 20 min at room temperature under the exclusion of light, and the success of the click reaction was directly analyzed by SDS-PAGE and UV visualization. For the semipreparative synthesis of conjugate 6, 2.5 mg of protein 3 and 1 mg of protein 5 were mixed and treated with TCEP, TBTA, and CuSO4 for 20 min as described earlier. Following the CuAAC reaction, EDTA (5 mM) was added to the solution and subsequently dialyzed against anion exchange buffer A (20 mM Tris, pH 9.3). Stock solutions were prepared as follows: 50 mM TCEP, 100 mM CuSO4, 500 mM EDTA in ddH2O each, and 1.7 mM TBTA in 1 : 4 (v/v) DMSO/tBuOH. For a kinetic analysis, proteins 3 and 5 were mixed at 20 μM concentration each, and the CuAAC reaction was started with the addition of CuSO4 as described earlier. The same reaction mixture without copper served as a negative control. Reactions were stopped by adding NaIO4 (0.4 mM), EDTA (0.5 mM), and neocuproine (0.5 mM) to the reaction mixture [24]. Samples were then run on an SDS-PAGE gel, and the band corresponding to the click product (6) was analyzed densitrometrically using the Scion Image software (Scion Corporation, Frederick, MD, USA). Experiments were performed in triplicate. RanGAP Pulldown Assays For GST-RanBP2 interaction studies, 75 μmol GST-RanBP2 and 75 μmol Ubc9 were incubated with 150 μmol of the respective interaction partners for 1.5 h on ice. The mixture was then applied to 25 μl GST-bind resin (Merck, Darmstadt, Germany) and incubated for 1 h at room temperature under gentle agitation. The beads were then washed three times with pulldown buffer before the proteins were eluted with pulldown buffer supplemented with 10 mM glutathione. Samples were analyzed by SDS-PAGE and western blot using SUMO1 pAb (kindly provided by Frauke Melchior, ZMBH Heidelberg) and His6 pAb (Roche, Mannheim, Germany).
Results and Discussion Amber Stop Codon Suppression Fails for Efficient AzF Incorporation into RanGAP1 RanGAP1 is one of the few proteins that can be efficiently SUMOylated in vitro using ATP and the E1 and E2 enzymes [38,40]. Nevertheless, it represents a good and challenging test case for our chemical conjugation approach because of its size,
availability of a native standard for comparison, and potential future applications in cell extracts or live cells. The SUMOylation site on human RanGAP1 is Lys524. We utilized a fragment consisting of aa 398–587 (His6-RanGAP1, 1) for expression in E. coli that is established for SUMOylation assays and its interaction with Ubc9 and RanBP2. Because this fragment contains a surface-exposed cysteine at position 573, we first tested our second click chemistry approach using AzF incorporation [25] to introduce the azide group (Scheme 1A, Approach 2). To this end, the expression plasmid for His-tagged RanGAP1 (aa 398–587) with an amber stop codon at position 524 was prepared by site-directed mutagenesis. E. coli BL21(DE3) cells were co-transformed with this plasmid and the plasmid encoding the aminoacyl-tRNA synthetase and its cognate amber suppressor tRNA, derived from the Methanococcus jannaschii TyrRS/tRNA pair, for the incorporation of AzF [30]. However, following the addition of the unnatural amino acid AzF and IPTG for induction of expression to the growth medium of these cells, only trace amounts of the desired full-length and azide-modified protein could be observed (data not shown), whereas control strains with previously reported amber suppression plasmids showed good expression levels under the same conditions (data not shown). It is well known that the amber suppression technology is dependent on the sequence context in the mRNA template. The suppression can be inefficient because of the competition with release-factor 1 (RF1), for example [41]. We therefore abandoned this strategy for the RanGAP1 protein. RanGAP1 Modification at a Single Cysteine in a K524C Mutant As the alternative route, we turned to our first click chemistry approach based on the modification of a single cysteine residue (Scheme 2). To ensure regioselectivity for the cysteine alkylation reaction, Cys573 in the human RanGAP1 fragment (His6-RanGAP1, 1) was first mutated to a serine to give the expression construct for His6-RanGAP1C573S (construct 2). Next, the acceptor Lys524 was mutated to cysteine in the expression plasmid for His6-RanGAP1C573S,K524C (construct 3). All three proteins were expressed in E. coli BL21(DE3) and purified to apparent homogeneity by Ni-NTA affinity chromatography. Next, construct 3 was reacted with iodoacetamide ethylazide [24] to give His6-RanGAP1N3 (4). Excess azide reagent was removed by dialysis. The synthesis of the alkyne-bearing His6-SUMO1(ΔGG)-PA (5) was carried out as previously described [24]. To confirm the presence of both azide and alkyne functionalities in proteins 4 and 5, CuAAC reactions were performed with alkyne-dansylamide and azide-tetramethylrhodamine, respectively. Both proteins could be efficiently labeled with the fluorophores (Figure 1). To generate the triazole-linked conjugate SUMO1(ΔGG) **RanGAP1 (6), proteins 4 and 5 were mixed in a 1 : 1 ratio
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Scheme 2. Preparation of the synthetic SUMO1**RanGAP1 conjugate.
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CLICK SUMOylation
Figure 1. Protein reagents modified with fluorophores using CuAAC. Shown are the reaction mixtures analyzed by SDS-PAGE under UV illumination and Coomassie staining. The presence or absence of the copper catalyst is indicated. His6-SUMO1(ΔGG)-Pa was reacted with azide-functionalized tetramethylrhodamine, and His6-RanGAP1N3 was incubated with alkyne-functionalized dansylamide (M = molecular weight marker).
(20 μM each) and incubated under click conditions with TCEP, TBTA, and CuSO4. Figure 2 shows the progress of this reaction monitored by SDS-PAGE analysis. After 15 min, about 40% of the starting materials were reacted to the desired conjugate 6, and the reaction progress reached a plateau. The rapid conjugate formation underlined once more the efficiency of the CuAAC reaction despite the macromolecular nature of the two reactants. Next, the reaction was performed on a semipreparative scale and stopped after 20 min by the addition of EDTA. Purification of 6 from the CuAAC reaction mixture was achieved by anion exchange chromatography [Figure S1 (see Supporting Information)]. Binding of SUMOylated RanGAP1 to RanBP/Ubc9 In order to biochemically characterize the chemical conjugate SUMO1(ΔGG)**RanGAP1 (6), we decided to perform a binding assay with RanBP2 and Ubc9, as previously reported [36]. For this purpose, we also prepared the natively modified SUMO1*RanGAP1 (7) as positive control and SUMO1*RanGAP1C573S (8) as a control for the potential influence of the Cys573Ser mutation. Both these conjugates were obtained by in vivo modification in E. coli cells that co-expressed the respective RanGAP1 proteins with SUMO1 and the E1 and E2 enzymes of the SUMO system [39]. Following cell lysis, the desired proteins were purified by Ni-NTA and anion exchange column chromatography [Figures S2 and S3 (see Supporting Information)]. Purified SUMOylated proteins and unconjugated control proteins were each mixed with GST-RanBP2 (aa 2553–2838) and Ubc9. Subsequently, the GST beads were added to the mixture, washed, and eluted with glutathione. Analysis of the eluted proteins showed that very similar amounts of the
chemical SUMO1**RanGAP1 conjugate 6 and the enzymatic SUMO1*RanGAP1 control conjugates 7 and 8 were retained on the column (Figures 3 and S4). In agreement with previous reports [33,36], no interaction could be observed under these conditions using unconjugated RanGAP1 or SUMO1. This result indicates that the triazole-linked chemical conjugate 6 behaved similarly to the isopeptide-linked native conjugates 7 and 8.
Conclusions In summary, we have described a new example for the chemical conjugation of the post-translational modifier SUMO to a substrate protein to give human SUMO1**RanGAP1. Our results lend further support to the general utility of the click chemistry approach to prepare such defined protein conjugates. Both SUMO1 and RanGAP1 were obtained from recombinant expression. Obviously, the employed click chemistry reactions including the use of copper ions did not denature or harm the proteins, as binding to the interaction partners RanBP2 and Ubc9 was not affected under the conditions tested. This work also underlines the importance of alternative routes to the incorporation of the functional group for bioorthogonal chemistry into the substrate protein, e.g. the azido moiety in RanGAP1 in this case. Unnatural amino acid mutagenesis to incorporate AzF, or other suitable amino acids like pyrrolysine derivatives [27], should in principle be applicable to any protein, and this technique would be compatible with the presence of cysteines. However, the suppression approach is technically more demanding and can lead to severely reduced levels of overexpressed proteins, as observed
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Figure 2. Click conjugation. (A) Reaction progress of the CuAAC reaction between His6-SUMO1(ΔGG)-Pa (5) and His6-RanGAP1N3 (4) monitored by Coomassie-stained SDS-PAGE. Reactions were quenched at the given time points by the addition of NaIO4, neocuproine, and EDTA. (B) Densitometric analysis of data shown in (A).
VAN TREEL AND MOOTZ
Figure 3. Formation of the RanBP2/Ubc9/SUMO1-RanGAP1 complex. SUMOylated proteins as well as RanGAP1 and SUMO1 controls as indicated were mixed with GST-RanBP2 (aa 2553–2838) and Ubc9 and incubated on ice for 1 h. Subsequently, the mixture was applied to GST beads, and the suspension was slightly agitated for 1 h at room temperature, followed by washing and elution of the beads using glutathione. Shown are western blots of an SDS-PAGE analysis of the elution fractions. 6 = SUMO1(ΔGG)**RanGAP1; 8 = SUMO1*RanGAP1C573S; 7 = SUMO1*RanGAP1; 4 = His6-RanGAP1N3; 2 = His6-RanGAP1C573S; 1 = His6-RanGAP1; and 5 = His6-SUMO1(ΔGG)-Pa. The calculated size of the SUMO1*RanGAP1 conjugate (7) is 33.9 kDa.
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here for RanGAP1. One possibility to circumvent this problem can be the use of RF1 knock-out strains [41]. On the other hand, the insertion of a single cysteine at the desired position for Ubl attachment is less likely to have an impact on expression levels of the protein of interest and is easy to perform. In this case, the removal of additional reactive cysteines must be tolerated by the protein, as we could show here for RanGAP1. Thus, both approaches outlined in Scheme 1 can be of practical relevance. The other point to be considered is the potential impact of the unnatural linker structure on the biochemical function of the protein conjugate. In fact, in most cases, the protein interactions brought about by conjugated Ubl modifiers like Ub and SUMO are mediated through specific binding interfaces on the surfaces of their globular structure that are remote from the isopeptide linkage, and the isopeptide linkage is not involved in the interaction surface. For Ub, the interaction hot spot is the hydrophobic patch around Ile44 [42,43], and for SUMO isoforms, it is a specific groove binding to peptide sequences with SUMO-interaction motifs [44]. Thus, changes in the linkage are likely tolerated as long as they provide similar distance and structural flexibility for the orientation of the Ubl. All reports on chemical conjugates with non-native linker structures so far generally support this view. On the other hand, it is clear that in some cases, a non-native structure, like the triazole linker discussed in this work, will have an influence on the structure and function of the proteins and their binding partners. Most notably, the enzymes involved in the deconjugation of the Ubl interact with the isopeptide bond [45,46]. Other examples are linkage-specific antibodies directed against Ub chains [47,48]. Interestingly, also in the complex SUMO1*RanGAP1/RanBP2/Ubc9 discussed here, a specific interaction of Ubc9 with the isopeptide bond can be observed in the crystal structure [35] but represents only a part of the interaction surface. Further studies will be required to reveal the nature of a potential direct interaction of Ubc9 with the non-native linker in the complex. In general, as long as the change imposed by the non-native linker is not too significant, chemical Ubl-protein conjugates may thus hold a great potential for future studies also in these cases.
Acknowledgements We thank Stefanie Sommer for discussions. We thank Frauke Melchior, Gerrit J. Praefcke, and Peter G. Schultz for providing plasmids. This work was funded by the Deutsche Forschungsgemeinschaft (SFB858).
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site.
127 J. Pept. Sci. 2014; 20: 121–127 Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci
Revised: 23 October 2013
Accepted: 24 October 2013
Published online in Wiley Online Library: 15 December 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2591
SUMOylated RanGAP1 prepared by click chemistry‡ Nadine D. van Treel and Henning D. Mootz* Ubiquitin and ubiquitin-like proteins such as SUMO represent important and abundant post-translational modifications involved in many cellular processes. These modifiers are reversibly attached via an isopeptide bond to lysine side chains of their target proteins by the action of specific E1, E2, and E3 enzymes. A significant challenge in studying ubiquitylation and SUMOylation is the frequently encountered inability to access desired conjugates at a defined position of the target protein and in homogenous form by using enzymatic preparation. In recent years, several chemical conjugation approaches have been developed to overcome this limitation. In this study, we aimed to selectively SUMOylate a 189-amino acid fragment of human RanGAP1 (amino acids 398–587) at the position of Lys524 by applying two recently reported approaches based on the Cu(I)-catalyzed alkyne-azide cycloaddition. Because of low yields observed for the incorporation of an unnatural amino acid with an azide moiety by the tRNA suppression technology, this route was abandoned. However, installing a single cysteine at position 524 and its selective alkylation was successful to introduce the azide group. The triazole-linked SUMO1**RanGAP1 conjugate could be obtained in good yields, purified, and was shown to specifically interact with RanBP2/Ubc9. Thus, we expand the scope of proteins accessible to chemical conjugation with ubiquitin-like proteins and underline the importance of having alternative approaches to do so. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: bioorthogonal; click chemistry; CuAAC; post-translational modification; protein chemistry; SUMO; ubiquitin
Introduction
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* Correspondence to: Henning D. Mootz, Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany. E-mail: henning. [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3–6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). Institute of Biochemistry, University of Muenster, Wilhelm-Klemm-Str. 2, 48149 Münster, Germany Abbreviations: aa, amino acids; AzF, p-azidophenylalanine; CuAAC, coppercatalyzed alkyne-azide cycloaddition; NTA, nitrilotriacetic acid; RF1, release-factor 1; SUMO, small ubiquitin-like modifier; TBTA, tris(benzyltriazolylmethyl)amine; TCEP, tris(2-carboxyethyl)phosphine; Ub, ubiquitin; Ubl, ubiquitin-like protein; LB, lysogeny broth; IPTG, isopropyl beta-D-1-thiogalactopyranoside; TB, transport buffer; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GST, glutathion S-transferase; PML, promyelocytic leukemia protein.
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
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The post-translational modification of proteins with ubiquitin (Ub) and Ub-like proteins (Ubls) such as small Ub-like modifier (SUMO) plays an important role in regulation of numerous processes in higher organisms, for example, proteasomal degradation, cell-cycle control, and regulation of signaling processes [1–3]. Ubls are typically attached to a specific lysine side chain of the target or substrate protein by an isopeptide bond involving their C-terminal carboxylate group. Ub and SUMO can also form chains by attachment of an additional post-translational modifier onto one of the seven lysines or the N-terminal amino group within Ub itself [4], or within Lys11 in the case of the human SUMO2/3 isoforms [5–7], respectively. The formation of Ubl conjugates is catalyzed by the action of dedicated E1, E2, and E3 enzymes and counteracted by specific isopeptidases, such as the deubiquitylases and the SUMO-deconjugating enzymes (e.g. the sentrin-specific protease proteins in humans), thereby rendering this process reversible [2,3,8]. Importantly, because the pattern of post-translational modification relies on the substrate specificity, activity, and localization of the conjugating enzymes and is not directly genetically encoded, it is impossible to predict Ubl conjugates with certainty or to obtain them by simple recombinant protein production. In fact, most conjugates cannot be prepared by enzymatic means at all, because not all E2 and E3 enzymes are sufficiently characterized and because enzymatic preparation can lead to mixtures and incomplete modifications at desired positions. However, in order to understand the biochemical, structural, and cellular consequences of Ubl modification, protein preparations with a defined attachment of the modifier at a desired position and in homogeneous form are required. For these reasons, significant research efforts have been
undertaken by several groups in recent years to develop nonenzymatic routes to ubiquitylated or SUMOylated proteins. Several chemical conjugation strategies have been reported that enable the preparation of modified proteins in homogeneous form with freely chosen attachment points [9–11]. Bond formation between the protein of interest and Ub/SUMO was achieved by alkylation [12], native and isopeptide chemical ligation [13–18], amide coupling [19], disulfide formation [20,21], thiol-ene addition [22], oxime ligation [23], and copper-catalyzed alkyne-azide cycloaddition (CuAAC, or click reaction) [24–27]. These approaches can be categorized into those yielding the native structure of the isopeptide bond and those giving rise to analogous linker structures. Furthermore, in some cases, the protein of interest is accessed by total chemical synthesis, whereas in others, it is obtained by recombinant protein expression.
VAN TREEL AND MOOTZ
Scheme 1. Two approaches for synthetic Ubl conjugates prepared by click chemistry. Ubl proteins can be ubiquitin and SUMO, for example. (A) Overview of the two approaches to incorporate the azido moiety into the recombinant substrate protein. CuAAC reaction with the C-terminally alkyne-modified Ubl modifier gives the desired conjugate. Note that either one or two of the conserved C-terminal glycines on the Ubl were deleted for approaches 2 and 1, respectively, to best resemble the length of the native isopeptide bond. (B) Preparation of the alkyne-modified Ubl by using an intein fusion protein to generate the reactive thioester (Pa = propargylamine).
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We [24–26] and others [27] have previously explored the CuAAC reaction [28,29] to attach the Ubl by a triazole linkage to the protein of interest. To this end, two approaches were developed by us (Scheme 1). In both cases, the C terminus of the Ubl was functionalized with an alkyne moiety by aminolysis of an intein thioester with propargylamine (Scheme 1B), but two different routes for incorporating the azide group were explored. In the first approach, the acceptor lysine in the substrate protein was mutated to a cysteine, and the thiol moiety of this amino acid was subsequently used to introduce the azide group for the click reaction [24]. In order to achieve regioselectivity, the introduced cysteine has to be the only cysteine in the substrate protein, or at least the only cysteine accessible and reactive under the conditions used. To circumvent this limitation, we developed the second approach (Scheme 1A) in which the azide group is introduced by unnatural amino acid mutagenesis [30,31] using the amber suppression technology [25,26]. Specifically, p-azidophenylalanine (AzF) was introduced at the desired position and could be used in the click reaction even in the presence of other cysteines in the same protein, as shown for the synthesis of a SUMO conjugate with the E2-conjugating enzyme Ubc9 [25]. For both approaches, we could show that the copper catalyst was not harmful as long as its exposure to the proteins was restricted to 30–60 min. Thus, the conjugation reactions could be performed under non-denaturing conditions. This point is important, because a key advantage of both approaches is that the substrate protein can be prepared recombinantly and thus does
not underlie any size restrictions that have to be considered in alternative fully synthetic approaches. Furthermore, the stability of both triazole linkers against hydrolytic cleavage by isopeptidases [24,26] holds a great potential for pulldown studies in cell extracts, for example. Our biochemical data reported so far on a PML-SUMO2, a Ubc9-SUMO2, and seven Ub–Ub conjugates and their interaction partners support the idea that both triazole linkers are good PML-SUMO2 [24], a Ubc9-SUMO2 [25], and seven Ub-Ub conjugates [26] and their structural mimics of the native isopeptide bond. This view could be further strengthened by QM/MM calculations for the triazole linker involving the unnatural amino acid AzF [32]. Ran GTPase activating protein (RanGAP1) was the first SUMOylated protein to be identified [33,34]. SUMO1 is one of the three human SUMO isoforms known to be conjugated to proteins. In contrast to the highly homologous SUMO2/3, SUMO1 does not form chains. SUMO1*RanGAP1 has been shown to be part of a stable pore complex with the Ran-binding protein RanBP2 at the nuclear envelope [33]. This interaction is enhanced by the presence of the E2-conjugating enzyme Ubc9, which exerts a synergistic effect on the complex formation [35,36]. It was shown that this complex forms an active SUMO E3 ligase [37]. The complex also plays an important role in transport processes across the nuclear membrane and in the cell cycle. Unmodified RanGAP1 and free SUMO1 molecules cannot interact with RanBP2 and fail to form the complex [33]. In this work, we aimed to further show the general applicability of the CuAAC conjugation approaches to other substrate proteins
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CLICK SUMOylation and synthesized a RanGAP1-SUMO1 conjugate. We found that the amber suppression technology to insert the unnatural amino acid AzF performed only very poorly for the RanGAP1 fragment of 189 amino acids (aa) in size. However, introducing the azide at the desired position via a cysteine was successful and underlines the versatility of the two CuAAC approaches for Ubl conjugation.
Materials and Methods General Standard molecular biology procedures were applied for DNA cloning and expression. Antibiotics were used at 100 μg/ml (ampicillin) and 30 μg/ml (chloramphenicol). Synthetic DNA oligonucleotides were purchased from Biolegio (Nijmegen, the Netherlands). All DNA vectors were confirmed by DNA sequencing (GATC Biotech, Konstanz, Germany). Site-directed mutagenesis was performed according to the QuickChange protocol from Stratagene. Cloning of Expression Plasmids pNW12 for the expression of His6-SUMO1(ΔGG)-GyrA-CBD was cloned as previously described [24]. pNW26 for the expression of His6-RanGAPC573S: Using site-directed mutagenesis, a serine codon was introduced at the position of the C573 codon with oligonucleotides 5′-CCCTGGAATCCTCGTCCTTCGCCCGC-3′ (forward; serine codon underlined) and 5′-GCGGGCGAAGGACGAGGATTCCAGGG-3′ (reverse) in the plasmid encoding for human RanGAP1 (aa 398– 587) [38]. pNW27 for expression of His6-RanGAPC573S,K524C: pNW26 served as a template for the site-directed mutagenesis to change the K524 codon to a cysteine codon using primers 5′CACATGGGTCTGCTCTGCAGTGAAGACAAGGTC-3′ (forward; cysteine codon underlined) and 5′-GACCTTGTCTTCACTGCAGAGCAGACCCATGTG-3′ (reverse). pNW35 for expression of His6-RanGAPK524AzF: An amber stop codon was inserted at the position of K524 in the abovementioned plasmid encoding human RanGAP1 using primers 5′-CACATGGGTCTGCTCTAGAGTGAAGACAAGGTC-3′ (forward; stop codon underlined) and 5′- GACCTTGTCTTCACTCTAGAGCAGACCCATGTG-3′ (reverse). Protein Expression and Purification
Cysteine Alkylation Construct 3 (His6-RanGAP1C573S,K524C; ~50 μM, pH 7.0) was treated for 20 min with 5 eq. tris(2-carboxyethyl)phosphine (TCEP), followed by addition of 20 eq. iodoacetamide ethyl azide [24]. After 10 min, again 5 eq. TCEP were added, and steps repeated twice. After 3.5 h total reaction time, 60 eq. DTT were added to quench excess iodoacetamide, and the protein was dialyzed against dialysis buffer (20 mM HEPES, 150 mM NaCl, pH 8.0).
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Protein expression was performed in Escherichia coli BL21(DE3) cells, which were transformed with the corresponding plasmids. All cells were grown in LB media supplemented with ampicillin at 37 °C to an OD600 of 0.6, and expression was induced with 0.4 mM IPTG and carried out for 4–5 h at 28 °C, unless otherwise noted. Expressions of GST-RanBP2 (GST explained in abbreviations) (aa 2553–2838, expression at 30 °C), Ubc9 (expression at 37 °C), and His6-RanGAP (aa 398–587) were performed using plasmids kindly provided by Frauke Melchior, ZMBH Heidelberg, Germany [38]. Coexpression of RanGAP1 proteins with SUMO1 was carried out in E. coli BL21(DE3) cells, which were co-transformed with the plasmids encoding for E1 and E2 enzymes as well as the native human His6-SUMO1 isoform (kindly provided by Gerrit Praefcke, University of Cologne, Germany) [39]. For expression of proteins with AzF [30], E. coli BL21(DE3) cells were co-transformed with the plasmid encoding the target gene containing the amber stop codon and the vector encoding the orthogonal pAzFRS/tRNACUA pair (kindly provided by Peter G. Schultz, Scripps Research Institute, La Jolla, USA). Cells were grown in M9-minimal media (45 mM Na2HPO4, 25 mM KH2PO4,
8.5 mM NaCl, 1 mM MgSO4, 0.1 mM CaCl2, 22 nM FeCl3, 0.03 mg/ml thiamine, 0.1% NH4Cl, 0.2% glucose) supplemented with 30 μg/ml chloramphenicol and 100 μg/ml ampicillin at 37 °C. When an OD600 of 0.6 was reached, AzF (purchased from Bachem, Bubendorf, Switzerland) was added to a final concentration of 1 mM, and protein expression was induced with 0.4 mM IPTG. Cultures were then incubated for another 4 h at 37 °C. All cells were harvested by centrifugation, resuspended in the respective buffer, and lysed by two passages through an EmulsiFlex-C5 (Avestin, Mannheim, Germany) emulsifier. In the case of Ubc9 expression, cells were harvested by centrifugation, resuspended in Ubc9 lysis buffer (50 mM Na2PO4, 50 mM NaCl, pH 6.5), and frozen at 80 °C. Thawing the cell pellet causes the protein to leak out of the cells, thus making cell lysis unnecessary and allowing for direct purification after removing cell debris by centrifugation [38]. Purification of chitin binding domain-tagged SUMO1 was carried out as previously described [24]. RanGAP1 proteins were purified on Ni-NTA agarose (Invitrogen, Darmstadt, Germany) equilibrated in Ni-NTA buffer A (50 mM Tris, 300 mM NaCl, pH 8.0) supplemented with 10 mM imidazole. After washing with Ni-NTA buffer A supplemented with 20 mM imidazole, a gradient was run ranging from 20 to 500 mM imidazole using an ÄKTA purifier (GE Healthcare, Munich, Germany). Cell lysate with GST-RanBP2 (aa 2553–2838) was incubated for 1 h at 4 °C with GST-bind resin equilibrated in RanBP2 lysis buffer [50 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA)], washed extensively with RanBP2 wash buffer [50 mM Tris–HCl, pH 8.0, 300 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT)], and then eluted using 20 mM glutathione in RanBP2 wash buffer. Fractions containing GST-RanBP2 were pooled and further purified by gel filtration using a Superdex 200 column (GE Healthcare, Munich, Germany) equilibrated in TB buffer [20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)2, 1 mM EGTA, 1 mM DTT, pH 7.3]. The purification of untagged Ubc9 [38] was performed via cation exchange chromatography at 4 °C using SP-sepharose (Fast Flow, Sigma-Aldrich, Schnelldorf, Germany) equilibrated in Ubc9 lysis buffer. After extensive washing with Ubc9 wash buffer (50 mM Na2PO4, 50 mM NaCl, 1 mM DTT, pH 6.5), the protein was eluted with Ubc9 elution buffer (50 mM Na2PO4, 300 mM NaCl, 1 mM DTT, pH 6.5). Fractions containing purified Ubc9 were pooled and dialyzed against TB buffer. All SUMO1-RanGAP1 conjugates were purified on Ni-NTA agarose as described earlier. After dialysis of the conjugates against anion exchange buffer A (20 mM Tris, pH 8.0), the conjugates were further purified by anion exchange chromatography by running a gradient from 0% to 100% buffer B (20 mM Tris, 500 mM NaCl, pH 8.0) in 30 min at a flow rate of 1 ml/min on a 1 ml MonoQ HP column (GE Healthcare, Munich, Germany). Proteins were then dialyzed in pulldown buffer [20 mM HEPES, 110 mM KOAc, 2 mM Mg(OAc)2, 1 mM EGTA and 0,05% Tween-20, pH 7.3] and stored at 80 °C. All protein concentrations were determined using the calculated absorbances at 280 nm.
VAN TREEL AND MOOTZ Cu(I) Catalyzed Azide-Alkyne Cycloaddition (CuAAC) [28,29] The fluorophores tetramethylrhodamine-azide and dansylamidealkyne (in DMF) were mixed in equimolar amounts (20 μM each) with corresponding reaction partners and treated with 500 μM TCEP. Then, tris(benzyltriazolylmethyl)amine (TBTA) at 50 μM and CuSO4 at 500 μM final concentrations were added to the solution. The CuAAC reaction was carried out for 20 min at room temperature under the exclusion of light, and the success of the click reaction was directly analyzed by SDS-PAGE and UV visualization. For the semipreparative synthesis of conjugate 6, 2.5 mg of protein 3 and 1 mg of protein 5 were mixed and treated with TCEP, TBTA, and CuSO4 for 20 min as described earlier. Following the CuAAC reaction, EDTA (5 mM) was added to the solution and subsequently dialyzed against anion exchange buffer A (20 mM Tris, pH 9.3). Stock solutions were prepared as follows: 50 mM TCEP, 100 mM CuSO4, 500 mM EDTA in ddH2O each, and 1.7 mM TBTA in 1 : 4 (v/v) DMSO/tBuOH. For a kinetic analysis, proteins 3 and 5 were mixed at 20 μM concentration each, and the CuAAC reaction was started with the addition of CuSO4 as described earlier. The same reaction mixture without copper served as a negative control. Reactions were stopped by adding NaIO4 (0.4 mM), EDTA (0.5 mM), and neocuproine (0.5 mM) to the reaction mixture [24]. Samples were then run on an SDS-PAGE gel, and the band corresponding to the click product (6) was analyzed densitrometrically using the Scion Image software (Scion Corporation, Frederick, MD, USA). Experiments were performed in triplicate. RanGAP Pulldown Assays For GST-RanBP2 interaction studies, 75 μmol GST-RanBP2 and 75 μmol Ubc9 were incubated with 150 μmol of the respective interaction partners for 1.5 h on ice. The mixture was then applied to 25 μl GST-bind resin (Merck, Darmstadt, Germany) and incubated for 1 h at room temperature under gentle agitation. The beads were then washed three times with pulldown buffer before the proteins were eluted with pulldown buffer supplemented with 10 mM glutathione. Samples were analyzed by SDS-PAGE and western blot using SUMO1 pAb (kindly provided by Frauke Melchior, ZMBH Heidelberg) and His6 pAb (Roche, Mannheim, Germany).
Results and Discussion Amber Stop Codon Suppression Fails for Efficient AzF Incorporation into RanGAP1 RanGAP1 is one of the few proteins that can be efficiently SUMOylated in vitro using ATP and the E1 and E2 enzymes [38,40]. Nevertheless, it represents a good and challenging test case for our chemical conjugation approach because of its size,
availability of a native standard for comparison, and potential future applications in cell extracts or live cells. The SUMOylation site on human RanGAP1 is Lys524. We utilized a fragment consisting of aa 398–587 (His6-RanGAP1, 1) for expression in E. coli that is established for SUMOylation assays and its interaction with Ubc9 and RanBP2. Because this fragment contains a surface-exposed cysteine at position 573, we first tested our second click chemistry approach using AzF incorporation [25] to introduce the azide group (Scheme 1A, Approach 2). To this end, the expression plasmid for His-tagged RanGAP1 (aa 398–587) with an amber stop codon at position 524 was prepared by site-directed mutagenesis. E. coli BL21(DE3) cells were co-transformed with this plasmid and the plasmid encoding the aminoacyl-tRNA synthetase and its cognate amber suppressor tRNA, derived from the Methanococcus jannaschii TyrRS/tRNA pair, for the incorporation of AzF [30]. However, following the addition of the unnatural amino acid AzF and IPTG for induction of expression to the growth medium of these cells, only trace amounts of the desired full-length and azide-modified protein could be observed (data not shown), whereas control strains with previously reported amber suppression plasmids showed good expression levels under the same conditions (data not shown). It is well known that the amber suppression technology is dependent on the sequence context in the mRNA template. The suppression can be inefficient because of the competition with release-factor 1 (RF1), for example [41]. We therefore abandoned this strategy for the RanGAP1 protein. RanGAP1 Modification at a Single Cysteine in a K524C Mutant As the alternative route, we turned to our first click chemistry approach based on the modification of a single cysteine residue (Scheme 2). To ensure regioselectivity for the cysteine alkylation reaction, Cys573 in the human RanGAP1 fragment (His6-RanGAP1, 1) was first mutated to a serine to give the expression construct for His6-RanGAP1C573S (construct 2). Next, the acceptor Lys524 was mutated to cysteine in the expression plasmid for His6-RanGAP1C573S,K524C (construct 3). All three proteins were expressed in E. coli BL21(DE3) and purified to apparent homogeneity by Ni-NTA affinity chromatography. Next, construct 3 was reacted with iodoacetamide ethylazide [24] to give His6-RanGAP1N3 (4). Excess azide reagent was removed by dialysis. The synthesis of the alkyne-bearing His6-SUMO1(ΔGG)-PA (5) was carried out as previously described [24]. To confirm the presence of both azide and alkyne functionalities in proteins 4 and 5, CuAAC reactions were performed with alkyne-dansylamide and azide-tetramethylrhodamine, respectively. Both proteins could be efficiently labeled with the fluorophores (Figure 1). To generate the triazole-linked conjugate SUMO1(ΔGG) **RanGAP1 (6), proteins 4 and 5 were mixed in a 1 : 1 ratio
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Scheme 2. Preparation of the synthetic SUMO1**RanGAP1 conjugate.
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CLICK SUMOylation
Figure 1. Protein reagents modified with fluorophores using CuAAC. Shown are the reaction mixtures analyzed by SDS-PAGE under UV illumination and Coomassie staining. The presence or absence of the copper catalyst is indicated. His6-SUMO1(ΔGG)-Pa was reacted with azide-functionalized tetramethylrhodamine, and His6-RanGAP1N3 was incubated with alkyne-functionalized dansylamide (M = molecular weight marker).
(20 μM each) and incubated under click conditions with TCEP, TBTA, and CuSO4. Figure 2 shows the progress of this reaction monitored by SDS-PAGE analysis. After 15 min, about 40% of the starting materials were reacted to the desired conjugate 6, and the reaction progress reached a plateau. The rapid conjugate formation underlined once more the efficiency of the CuAAC reaction despite the macromolecular nature of the two reactants. Next, the reaction was performed on a semipreparative scale and stopped after 20 min by the addition of EDTA. Purification of 6 from the CuAAC reaction mixture was achieved by anion exchange chromatography [Figure S1 (see Supporting Information)]. Binding of SUMOylated RanGAP1 to RanBP/Ubc9 In order to biochemically characterize the chemical conjugate SUMO1(ΔGG)**RanGAP1 (6), we decided to perform a binding assay with RanBP2 and Ubc9, as previously reported [36]. For this purpose, we also prepared the natively modified SUMO1*RanGAP1 (7) as positive control and SUMO1*RanGAP1C573S (8) as a control for the potential influence of the Cys573Ser mutation. Both these conjugates were obtained by in vivo modification in E. coli cells that co-expressed the respective RanGAP1 proteins with SUMO1 and the E1 and E2 enzymes of the SUMO system [39]. Following cell lysis, the desired proteins were purified by Ni-NTA and anion exchange column chromatography [Figures S2 and S3 (see Supporting Information)]. Purified SUMOylated proteins and unconjugated control proteins were each mixed with GST-RanBP2 (aa 2553–2838) and Ubc9. Subsequently, the GST beads were added to the mixture, washed, and eluted with glutathione. Analysis of the eluted proteins showed that very similar amounts of the
chemical SUMO1**RanGAP1 conjugate 6 and the enzymatic SUMO1*RanGAP1 control conjugates 7 and 8 were retained on the column (Figures 3 and S4). In agreement with previous reports [33,36], no interaction could be observed under these conditions using unconjugated RanGAP1 or SUMO1. This result indicates that the triazole-linked chemical conjugate 6 behaved similarly to the isopeptide-linked native conjugates 7 and 8.
Conclusions In summary, we have described a new example for the chemical conjugation of the post-translational modifier SUMO to a substrate protein to give human SUMO1**RanGAP1. Our results lend further support to the general utility of the click chemistry approach to prepare such defined protein conjugates. Both SUMO1 and RanGAP1 were obtained from recombinant expression. Obviously, the employed click chemistry reactions including the use of copper ions did not denature or harm the proteins, as binding to the interaction partners RanBP2 and Ubc9 was not affected under the conditions tested. This work also underlines the importance of alternative routes to the incorporation of the functional group for bioorthogonal chemistry into the substrate protein, e.g. the azido moiety in RanGAP1 in this case. Unnatural amino acid mutagenesis to incorporate AzF, or other suitable amino acids like pyrrolysine derivatives [27], should in principle be applicable to any protein, and this technique would be compatible with the presence of cysteines. However, the suppression approach is technically more demanding and can lead to severely reduced levels of overexpressed proteins, as observed
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Figure 2. Click conjugation. (A) Reaction progress of the CuAAC reaction between His6-SUMO1(ΔGG)-Pa (5) and His6-RanGAP1N3 (4) monitored by Coomassie-stained SDS-PAGE. Reactions were quenched at the given time points by the addition of NaIO4, neocuproine, and EDTA. (B) Densitometric analysis of data shown in (A).
VAN TREEL AND MOOTZ
Figure 3. Formation of the RanBP2/Ubc9/SUMO1-RanGAP1 complex. SUMOylated proteins as well as RanGAP1 and SUMO1 controls as indicated were mixed with GST-RanBP2 (aa 2553–2838) and Ubc9 and incubated on ice for 1 h. Subsequently, the mixture was applied to GST beads, and the suspension was slightly agitated for 1 h at room temperature, followed by washing and elution of the beads using glutathione. Shown are western blots of an SDS-PAGE analysis of the elution fractions. 6 = SUMO1(ΔGG)**RanGAP1; 8 = SUMO1*RanGAP1C573S; 7 = SUMO1*RanGAP1; 4 = His6-RanGAP1N3; 2 = His6-RanGAP1C573S; 1 = His6-RanGAP1; and 5 = His6-SUMO1(ΔGG)-Pa. The calculated size of the SUMO1*RanGAP1 conjugate (7) is 33.9 kDa.
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here for RanGAP1. One possibility to circumvent this problem can be the use of RF1 knock-out strains [41]. On the other hand, the insertion of a single cysteine at the desired position for Ubl attachment is less likely to have an impact on expression levels of the protein of interest and is easy to perform. In this case, the removal of additional reactive cysteines must be tolerated by the protein, as we could show here for RanGAP1. Thus, both approaches outlined in Scheme 1 can be of practical relevance. The other point to be considered is the potential impact of the unnatural linker structure on the biochemical function of the protein conjugate. In fact, in most cases, the protein interactions brought about by conjugated Ubl modifiers like Ub and SUMO are mediated through specific binding interfaces on the surfaces of their globular structure that are remote from the isopeptide linkage, and the isopeptide linkage is not involved in the interaction surface. For Ub, the interaction hot spot is the hydrophobic patch around Ile44 [42,43], and for SUMO isoforms, it is a specific groove binding to peptide sequences with SUMO-interaction motifs [44]. Thus, changes in the linkage are likely tolerated as long as they provide similar distance and structural flexibility for the orientation of the Ubl. All reports on chemical conjugates with non-native linker structures so far generally support this view. On the other hand, it is clear that in some cases, a non-native structure, like the triazole linker discussed in this work, will have an influence on the structure and function of the proteins and their binding partners. Most notably, the enzymes involved in the deconjugation of the Ubl interact with the isopeptide bond [45,46]. Other examples are linkage-specific antibodies directed against Ub chains [47,48]. Interestingly, also in the complex SUMO1*RanGAP1/RanBP2/Ubc9 discussed here, a specific interaction of Ubc9 with the isopeptide bond can be observed in the crystal structure [35] but represents only a part of the interaction surface. Further studies will be required to reveal the nature of a potential direct interaction of Ubc9 with the non-native linker in the complex. In general, as long as the change imposed by the non-native linker is not too significant, chemical Ubl-protein conjugates may thus hold a great potential for future studies also in these cases.
Acknowledgements We thank Stefanie Sommer for discussions. We thank Frauke Melchior, Gerrit J. Praefcke, and Peter G. Schultz for providing plasmids. This work was funded by the Deutsche Forschungsgemeinschaft (SFB858).
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Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site.
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