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Expeditious Chemical Synthesis of Ubiquitinated Peptides Employing Orthogonal Protection and Native Chemical Ligation K. S. Ajish Kumar, Liat Spasser, Shimrit Ohayon, Lesly A. Erlich, and Ashraf Brik* Department of Chemistry, Ben Gurion University, Beer Sheva, Israel 84105
bS Supporting Information ABSTRACT: Ubiquitination;the attachment of ubiquitin to a protein target;is involved in a wide range of cellular processes in eukaryotes. This dynamic posttranslational modification utilizes three enzymes to link, through an isopeptide bond, the C-terminal Gly of ubiquitin to the lysine side chain from a protein target. Progress in the field aiming at deciphering the role of ubiquitination in biological processes has been very dependent on the discovery of the enzymatic machinery, which is known to be very specific to each protein target. Chemical approaches offer a complementary route to the biochemical methods to construct these conjugates in vitro in order to assist in unraveling the role of ubiquitination on protein function. Herein is presented a novel method for the rapid synthesis of ubiquitinated peptides employing solid-phase peptide to generate the critical isopeptide linkage. Using these tools, several ubiquitinated peptides derived from known ubiquitinated proteins were prepared. Among them is the ubiquitinated C-terminal fragment of H2B, which can be used in the synthesis of monoubiquitinated H2B. For the first time, we systematically assessed the effect of the length of the ubiquitinated peptides on the UCH-L3 activity and found that peptides of up to ∼20 residues are preferred substrates.
stable ubiquitinated conjugates.3-9 While these methods facilitated the synthesis of ubiquitinated peptides and proteins for a variety of important studies, the absence of a native and reversible isopeptide bond precludes the use of these conjugates to investigate the mechanisms by which the dynamics of ubiquitination (i.e., ubiquitination-deubiquitination) regulate protein function and biological processes.10,11 Site-specific ubiquitination of peptides and proteins via formation of a native isopeptide bond is now possible because of the work of several groups, including ours (Scheme 1). The first was introduced by Muir and co-workers, who relied on the use of a photocleavable auxiliary.12 Our group introduced the δ-mercaptolysine to promote isopeptide formation.13 Similarly, Liu's group devised the γ-mercaptolysine analogue to assist isopeptide formation in addition to backbone ligation.14 More recently, Chin and Komander developed genetically encoded orthogonal protection and activated ligation in the presence of AgNO3/HOSu in DMSO to generate the isopeptide linkage in a site-specific manner.15 In these studies, multistep syntheses (8-19 steps) are needed to prepare the desired auxiliary or the thiolysine analogues. Nevertheless, these strategies must be included in the synthetic scheme when attempting the chemical and semisynthesis of certain protein targets wherein the isopeptide must be formed after the assembly of the full-length protein.16-18 On the other hand, in some other cases such as the synthesis of ubiquitinated peptides, the process could be tedious and very limited to
C
hemical synthesis of ubiquitinated peptides and proteins offers an excellent opportunity to prepare these bioconjugates due to the precise control over the ubiquitination site and the large quantities that could be obtained of the desired product. Moreover, chemical methods are obviated from the use of conjugating enzyme, E2, and ubiquitin-protein ligase, E3, that are known to be very specific to each protein target.1 In addition to the E1 enzyme, which activates ubiquitin (Ub) C-terminus acid, these enzymes constitute the enzymatic machinery that is used to form the isopeptide bond between the ε-amine of a lysine residue from the target protein and the C-terminus of Ub.1 In a similar mechanism, other ubiquitin like modifiers (Ubl) are also tagged to a protein target leading to a variety of biochemical processes such as DNA-damage response, cell-cycle process, and a regulation of cell survival.2 As a result, protein modification with Ub or Ubl is a very complex posttranslational event and progress in the field aiming at deciphering the role of ubiquitination on biological processes has been very dependent on the discovery of the specific E2-E3 enzymatic machinery. Thus, there is an urgent need for efficient and reliable methods to generate sufficient amounts of a highly homogeneous ubiquitinated peptide and protein reagents. Chemical approaches offer a complementary route to the biochemical methods to construct these bioconjugates to assist in revealing the role of these modifications in cellular functions. These reagents could be used in assays, antigens for developing linkage-specific antibodies, and substrates for further enzymatic elaboration to elongate the Ub chain, which is linked to a peptide or protein target. Recently, several groups have introduced elegant chemical methods for linking Ub and Ubl to peptides or proteins via non-native isopeptide bonds to generate enzymatically r 2011 American Chemical Society
Received: October 27, 2010 Revised: December 23, 2010 Published: January 14, 2011 137
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Scheme 1. Current Methods Used to Form the Isopeptide Bond Assisted by (A) δ-Mercaptolysine, (B) γ-Mercaptolysine, (C) Photolabile Auxiliary, (D) Genetically Encoded Orthogonal Protection and Activated Ligation in the Presence of AgNO3/HOSu in DMSOa
a
AA is for amino acid, K* is for the modified Lys, R = -CH3, R0 = -(CH2)3CONH-CH3.
the synthetic community, rendering these methods inaccessible to many laboratories. Here, we report a novel method for the expeditious synthesis of ubiquitinated peptides, with native isopeptide bond, relying only on solid-phase peptide synthesis (SPPS) coupled with native chemical ligation (NCL).19 Using these methods, several ubiquitinated peptides derived from p53, di-Ub, and H2B were prepared. The latter ubiquitinated peptide is a key precursor in the synthesis of monoubiquitinated H2B. Moreover, a small set of ubiquitinated peptides derived from the C-terminal of H2B was also prepared to evaluate the effect of substrate length on the ubiquitin C-terminal hydrolase (UCH-L3) enzyme activity to hydrolyze the isopeptide bond. Our strategy for the rapid synthesis of ubiquitinated peptides is depicted in Scheme 2. In this approach, SPPS is used to build the target peptide linked through an isopeptide bond to the C-terminal fragment of Ub (46-76), 1. This peptide also bears N-terminal Cys instead of the original Ala46 residue, which is introduced to facilitate the NCL step with Ub(1-45) thioester 2. After the ligation step between peptides 1 and 2, to give the fulllength peptide-Ub 3, Cys46 will be converted to the native Ala46 applying well-established desulfurization conditions20 to furnish the unmodified ubiquitinated peptide 4.
To implement the above-described strategy, we designed a synthetic approach for the branched peptide, the most challenging precursor in our strategy. In order to accomplish this key step, we chose the model peptide LYKAG wherein the orthogonally protected Lys, Fmoc-Lys-(ivDde)-OH (ivDde = 1-[4,4dimethyl-2,6-dioxo-cyclohexylidene]-3-methylbutyl), is used to introduce the isopeptide in a site-specific manner (Scheme 3).21 The use of ivDde over the Dde protecting group has been shown to be superior, as this group is more stable under the conditions of Fmoc removal.22 After Fmoc-SPPS of peptide 5 (Scheme 3), the ε-amine of the Lys residue was unmasked selectively by applying 5% hydrazine in DMF, followed by Fmoc-SPPS of the Ub(46-76) segment. Side-chain deprotection and cleavage from the resin (TFA:H2O:TIS), followed by ether precipitation, revealed that peptide 11, without any further optimization, was efficiently synthesized as indicated by the crude peptide analysis using HPLC and mass spectrometry (Supporting Information). Preparative HPLC purification and lyophilization steps afforded the pure peptide 11 in 25% yield. The synthesis of the Ub(1-45)thioester 2 was carried out efficiently as we previously described,23 by applying Fmoc-SPPS and the N-acyl urea based chemistry.24 With these peptides in hand, we then turned our attention to the full assembly of the desired ubiquitinated peptide using NCL. 138
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the N- or C-termini. To expand the length of the backbone LYKAG peptide at the N-terminus, peptide 15 was prepared from 9 (Thz-LYK(ivDde)AG). Subsequent to the first ligation, the Thz was converted fully to Cys using methoxylamine27 to allow for sequential backbone ligation with LYRAG-SR, after which a desulfurization step (Scheme 3, path B) gave the ubiquitinated peptide 21 in 25% isolated yield over two steps (Supporting Information). In principle, one could also equip the C-terminus of the backbone peptide with a N-S acyl transfer device,28 which could be activated after the first ligation step furnishing the thioester functionality. Alternatively, a kinetically controlled ligation could be used to increase the length of the ubiquitinated peptide at the C-terminus. In this case, the backbone peptide would bear a less reactive alkylthioester compared to the thioester of peptide 2.29,30 Our successes with the rapid synthesis of several ubiquitinated peptides coupled with backbone sequential ligation prompted us to attempt the synthesis of ubiquitinated peptide derived from the C-terminal of H2B (H2B117-125). Peptide 25 with the photolabile auxiliary attached to Lys120 and bearing N-terminal Cys (Ala118Cys) was prepared previously by Muir and coworkers to allow for site-specific ubiquitination, followed by auxiliary and Cys protecting group removal to afford ubiquitinated peptide 26 (Scheme 4A). Subsequently, this has permitted backbone ligation with expressed H2B(1-117)-thioester, and a final desulfurization step enabled the semisynthesis of full-length monoubiquitinated H2B (Scheme 4A).31 One major noticeable obstacle in this work was the ubiquitination step, which proceeded slowly and required five days to achieve a moderate yield of ubiquitinated peptide 26. Our initial attempts to synthesize ubiquitinated H2B(118-125) 27, with N-terminal Thz employing a similar strategy to the previously synthesized ubiquitinated peptides (Scheme 3), were challenged by severely incomplete couplings of several residues from the branched C-terminal Ub peptide. We were unable to achieve a successful synthesis of this crucial fragment, and trials to change coupling reagents, resin type, loading of resin, and the use of microwave-assisted peptide coupling did not lead to a significant improvement. Since the backbone peptide H2B(118125) was the only difference compared to our previously synthesized ubiquitinated peptides 17-19, we hypothesized that the conformational behavior of the H2B backbone peptide might be the reason for the difficult couplings and the observed side reactions. To examine this, we used the pseudoproline32 derivative of Tyr121Thr122 as a building block to prevent any aggregation on resin, which could hinder efficient synthesis. Pleasantly, following these changes in our strategy we were able to achieve a highly efficient synthesis of this peptide (Supporting Information), which was isolated in 35% yield. Ligation with the Ub(1-45) thioester 2, followed by Thz to Cys conversion, gave the desired building block 28, in 31% yield. Conversion of this key ubiquitinated peptide to the full-length H2B could be preformed following the same procedure reported by Muir and co-workers (Scheme 4A). Our presented method should also enable rapid preparation of deubiquitinase (DUB) substrate libraries, which could shed light on the unique specificities of particular DUBs.9 This knowledge would assist in the discovery of specific inhibitors of important DUBs involved in health and disease.33 One such enzyme is the UCH-L3 enzyme, which catalyzes the removal of adducts from the C-terminus of Ub.34 Although most of these studies have
Scheme 2. General Strategy for the Synthesis of Ubiquitinated Peptidesa
a
The sequence of Ub and the ligation site is also shown. Met1 was replaced by a highly conservative modification Nle to avoid oxidation.
The ligation between peptides 2 and 11 (Scheme 3, path a) was carried out under NCL conditions, i.e., 6 M Gn 3 HCl, 200 mM phosphate buffer, pH 7.5, in the presence of 2% (v/v) thiophenol/benzylmercaptan. The reaction was followed by HPLC and mass spectrometry, which indicated nearly complete ligation after 12 h (Figure 1). Purification and lyophilization steps gave the desired product in 35% yield. The purified ligated product was subjected to metal-free desulfurization conditions,25,26 affording the unmodified branched ubiquitinated LYKAG, 17, in 66% yield, after 4 h (Figure 1). The desulfurized product 17 was folded and treated with UCH-L3 in which after 3 h a complete hydrolysis was achieved affording both the hydrolyzed Ub-COOH and the LYKAG peptide (Supporting Information). This study provides further evidence of the structural integrity of the isopeptide linkage and the correct folding of the Ub. Using the above-described strategy for the rapid synthesis of ubiquitinated peptides, two other ubiquitinated peptides, 18 and 19, which were derived from p53 and Lys-48 linked di-Ub, respectively, were also prepared in high efficiency (Supporting Information). Their preparation started from peptides 6 and 7, which were transformed to peptides 12 and 13 and through a sequence of reactions shown in Scheme 3 (path A) were converted to the ubiquitinated peptides 18 and 19, respectively. Not only should our approach allow the synthesis of short ubiquitinated peptides, but through further elaboration, one could also increase the length of the ubiquitinated peptide at 139
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Scheme 3. Synthetic Strategy for the Ubiquitinated Peptides 17-20 Employing the ivDde Orthogonal Protecting Group NCL and Desulfurization (Path a) and Ubiquitinated Peptides 21-24 Using Sequential Ligation (Path b)a
a
Nbz is for N-acyl benzimidazolinone functionality and Thz is for 1,3-thiazolidine 4-carboxo group. When peptide II is LYRAG, we employed Boc-SPPS.
been carried out on R-linked peptide to Ub,35,36 the general conception is that the UCH-L3 preferred substrates are Ub linked to small adducts such as a single amino acid, ethyl ester, and short peptides. Biochemical and structural analysis revealed that this enzyme and other members of this family use a disordered active site crossover loop of 20 residues, which imposes substrate filtering and restricts access of larger substrates.37-40 With regard to substrates that consist of Ub-linked to peptides, it has been suggested that UCH-L3 cleaves peptide extensions comprising up to 20 amino acids with high efficiency and low sequence preference. However, this hypothesis has not been fully addressed experimentally, and only one ubiquitinated peptide comprising 13 amino acids with a native isopeptide bond, which was enzymatically prepared and found to be a substrate for UCHL3.39 This is mainly because of the difficulties in preparing highly
homogeneous ubiquitinated peptides using the E1-E3 enzymatic machinery.33 To evaluate the effect of the substrate length on the UCH-L3 activity, we assembled four different ubiquitinated peptides (Scheme 3) derived from the C-terminal of H2B, 20-24 with various lengths (8, 15, 21, and 31 residues, respectively). These peptides were then tested under the same hydrolysis conditions with UCH-L3. The hydrolysis reaction was monitored by HPLC following the disappearance of the ubiquitinated peptide and the appearance of products, Ub-COOH and the H2B peptide (Figure 2). As seen in Figure 2, the peptides 20, 22, and 23 with up to 21 amino acids were cleaved with a similar efficiency and gave a 65-75% hydrolysis within 30 min and were completely disassembled within 90 min. However, when compared to the shorter peptides, the ubiquitinated peptide 24 comprising 31 residues 140
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Figure 1. Analytical HPLC trace/(ESIMS) of the ligation reaction between peptides 2 (1.3 equiv) and 11 in the presence of 2% (v/v) thiophenol/benzylmercaptan. Reported mass is for total protein. (A) Ligation at 0 h: Peak a corresponds to peptide 11 with the observed mass of 4034 Da (calcd mass = 4033.6 Da). Peak b corresponds to peptide 2 with the observed mass 5198 Da (calcd mass = 5198.9 Da). (B) Ligation at 12 h: peak c corresponds to remains of peptide 2; peak d corresponds to the ligation product with the observed mass of 9112.9 Da (calcd mass = 9111.5 Da). Ligation was carried out in 6 M Gn 3 HCl, 200 mM phosphate buffer, pH 7.5, in the presence of 2% (v/v) thiophenol/ benzylmercaptan and the product was isolated in 42% yield. (C) Desulfurization of the ligation product after 4 h: Peak e corresponds to the desired desulfurized product 17 with the observed mass of 9079.8 Da (calcd mass = 9079.5 Da). Peak * corresponds to thiol additives.
Figure 2. Evaluation of the UCH-L3 activity with ubiquitinated peptides 20 and 22-24: (A) HPLC and mass spectrometry analysis of the enzymatic cleavage of peptide 20 after 30 min. Peak a corresponds to the 8-mer peptide from H2B with the observed mass of 895.5 Da (calcd. 896.0 Da); peak b is the remaining starting material, peptide 20, with the observed mass of 9426.7 Da (calcd mass = 9425.8 Da); the major peak corresponds to Ub-COOH with the observed mass of 8549.0 Da (calcd mass = 8548.5 Da). (B) HPLC and mass spectrometry analysis of the enzymatic cleavage of peptide 22 after 30 min. Peak c corresponds to the 15-mer peptide from H2B with the observed mass of 1568.4 Da (calcd mass = 1568.8 Da); Ppak d is the remaining starting material, peptide 22, with the observed mass of 10100.9 Da (calcd mass = 10 098.7 Da), the major peak corresponds to Ub-COOH. (C) HPLC and mass spectrometry analysis of the enzymatic cleavage of peptide 23 after 30 min. Peak e corresponds to the 21-mer peptide from H2B with the observed mass of 2205.0 Da (calcd mass = 2204.5 Da); peak f is remaining starting material, peptide 23, with the observed mass of 10 735.9 Da (calcd mass = 10 734.3 Da); the major peak corresponds to Ub-COOH. (D) HPLC and mass spectrometry analysis of the enzymatic cleavage of peptide 24 after 30 min. Peak g corresponds to the 31-mer peptide from H2B with the observed mass of 3310.5 Da (calcd mass = 3309.9 Da); peak h is the starting material, peptide 24, with the observed mass of 11 841.4 Da (calcd mass = 11 839.8 Da). (E) The percent hydrolysis of ubiquitinated peptides 20 and 22-24, which was determined under same reaction conditions. Error bars correspond to the standard deviation of three measurements.
was hydrolyzed less efficiently affording 25% hydrolysis within 30 min and required 5 h for complete hydrolysis. These results support the notion that UCH-L3 tolerates various peptide sequences; however, it shows high sensitivity to the length of the peptide. In summary, we have shown a novel method for the rapid synthesis of ubiquitinated peptides employing only SPPS and NCL/desulfurization. Using these tools, several ubiquitinated peptides were straightforwardly prepared. One of these peptides is the ubiquitinated C-terminal H2B, which could lead to an efficient synthesis of monoubiquitinated H2B. We were able, for the first time, to assess the effect of the length of the ubiquitinated peptides on the UCH-L3 activity and found that peptides with up to ∼20 residues are the preferred substrates. Our method allows for full control of the ubiquitinated peptide, which could aid in studies of different Ub modifications, e.g., specific labeling. We believe that this approach should enable the rapid assembly of a variety of ubiquitinated peptides for various studies related to Ub 141
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Scheme 4. Preparation of Mono-Ubiquitinated H2Ba
a
(A) Muir approach for the semisynthesis of mono-ubiquitinated H2B employing a photolabile auxiliary, (B) alternative synthesis of monoubiquitinated H2B employing our strategy developed to prepare the key ubiquitinated peptide H2B(118-125). The sequence of the H2B is shown at the top, the ligation site is underlined, and K* corresponds to the ubiquitination site.
biology and facilitate the studies in unraveling the effect of ubiquitination on histone biology.
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’ ASSOCIATED CONTENT
bS
Supporting Information. Experimental procedures and characterization of the products and other detailed results. This material is available free of charge via the Internet at http://pubs. acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Fax (þ) 08-647-2944, Tel (þ) 08-6461195; E-mail:
[email protected].
’ ACKNOWLEDGMENT We are grateful to the Edmond J. Safra Foundation for financial support. 142
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