Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer Ling Xu,†,‡,∥ Jian-Feng Huang,‡,∥ Chen-Chen Chen,‡ Qian Qu,† Jing Shi,*,‡ Man Pan,§ and Yi-Ming Li*,† †

School of Biological and Medical Engineering, Hefei University of Technology, Hefei, Anhui 230009, China Department of Chemistry, School of Life Science, University of Science and Technology of China, Hefei 230026, China. § Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, United States ‡

S Supporting Information *

ABSTRACT: An efficient method for the assembly of polyUb chains using auxiliarymodified Ub isomers is reported. This strategy takes advantages of auxiliary-mediated native chemical ligation between the distal Ub C-terminal hydrazide and the auxiliary of proximal Ub. Using removable protecting groups, Lys48-linked and Lys6-linked tri-Ub and even a mixed-linkage Lys6, Lys48-linked triUb in multimilligram quantities was made. These results demonstrate that this strategy yields natural polyubiquitin chains of desired length and linkage by using Ub isomer.

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from simple operation and allows the performance of costeffective isotopic labeling in any monomer of the polyUb chain for structural analysis. However, silver-mediated thioster−amine condensation reaction needs to be performed under the blocking of all other primary amines by an allyloxycarbonyl (Alloc) or carbobenzyloxy (Cbz) protecting group, followed by removal of protecting group as necessary. The multistep operation and harsh ligation conditions result in relatively low yields of final product. Thus, the development of a new bacterially expressed Ub building block which makes the ligation and deprotection processes cost-effective is needed for the acquisition of polyUb chains. Herein, we report an alternative strategy using a premade auxiliary ((1-(2,4-dimethoxyphenyl)-2-mercaptoethyl)glycine)modified Ub isomer as a building block to synthesize the polyUb chain (Scheme 1C). The building block can be readily prepared through three steps: (1) genetically encoding Nε-propargyloxycarbonyl-L-lysine (Proc-Lys) at the designed site;9 (2) introducing the hydrazide group at the C-terminus of the Ub; and (3) installing the auxiliary that is capable of performing native chemical ligation (NCL) at Lys residue.6a,10 The Ub isomer bears a site-specific auxiliary group and a C-terminal hydrazide so that polyUb chains can be efficiently assembled through sequential hydrazide-based auxiliary-mediated NCL.11 The practicality of the strategy is exemplified by the preparation of Lys48-linked tri-Ub (denoted as K48-Ub3) chain and Lys6linked tri-Ub (K6-Ub3) chain, even a mixed-linkage Lys48, Lys6linked tri-Ub (K6,48-mixed Ub3) chain. We first attempted to synthesize this building block for K48linked polyUb chains (Figure 1A). To this end, Proc-Lys was genetically incorporated at residue 48 of a Ub variants Ub(1-

s one of the most important post-translational modifications in eukaryotic cells, polyubiquitination of proteins plays key roles in a series of physiological and disease-related processes, such as protein degradation, cellular immune response, and DNA damage repair.1 The major reason for the broad spectrum of regulation is that through Lys residues, ubiquitin (Ub) can form types of homogeneous polyubiquitin (polyUb) chains (M1, K6, K11, K27, K29, K33, K48, K63) as well as heterogeneous polyUb (i.e., mixed or branched chain).2 The distinct functional outcome of polyubiquitination of proteins mostly depends on the length and type involved in the Ub linkage. For example, in the case of the K48 linkage, tetraUb is necessary for the efficient substrate recognition by the 26S proteasome.3 On the other hand, the conformational differences between K48- and K63-linked chains are responsible for their distinct ability.4 However, a bottleneck for elucidating the molecular mechanisms of polyUb signals lies in the lack of polyUb chains with the native connectivity, controlled length, defined linkage, and sufficient (milligram scale) quantities. A variety of strategies for obtaining polyUb chains have been developed.5 Enzymatic methods can be used to prepare K48and K63-linked polyUb chains efficiently, but the synthesis of atypical Ub chains remains difficult. Nonenzymatic methods are mostly based on a premade modified Ub building block, and the polyUb chain is assembled through sequential ligations. Among them, types of polyUb chains can be produced through the direct formation of the isopeptide bond or the use of auxiliarymodified Lys residues (Scheme 1A), including γ/δ-mercaptolysine and glycyl-auxiliary-modified lysine.6 Although a remarkable accomplishment, the requirement to use total chemical synthesis has limited their utility in biochemical laboratory settings.7 Taking advantage of the bacterially expressed Ub building block in the assembly of polyUb chain, a genetically encoded orthogonal protection and activated ligation (GOPAL) method was developed (Scheme 1B).7,8 This strategy benefits © XXXX American Chemical Society

Received: November 13, 2017

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DOI: 10.1021/acs.orglett.7b03515 Org. Lett. XXXX, XXX, XXX−XXX

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Scheme 1. Schematics on the Assembly of polyUb Chains. (A) Mercaptolysine-Assisted Total Chemical Synthesis and Isopeptide Chemical Ligation. (B) Genetically Encoded Orthogonal Protection and Activated Ligation. (C) Genetically Encoded New Orthogonal Protection and Auxillary-Mediated Ligation

Ub(1-G76C) mutant is used because the C-terminus Cys of Ub(1-G76C) can be converted into the corresponding hydrazide via the N−S acyl transfer strategy.14 The recombinant expression of Ub(1-G76C)/K48Proc-Lys 3 was carried out similarly to that previously reported.9 Perchloric acid was then added to the bacterial lysate for precipitation of nontarget proteins.10 After centrifugation, the supernatant was concentrated and dialyzed to 50 mM Tris−HCl buffer. Subsequently, the hydrazinolysis reagent (Mesna 100 mg/mL, NH2NH2·HCl 50 mg/mL, tris(2-chloroethyl) phosphate 5 mg/mL, pH 7.0) was added to the supernatant, and the C-terminal cysteine of Ub could be converted into a hydrazide by an N−S acyl transfer reaction. High-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) analysis indicated that hydrazinolysis of a Gly−Cys motif proceeded smoothly and almost 80% protein 3 was converted into protein 4 (Ub(1−75)-NHNH2/K48Proc-Lys) at 37 °C after 54 h. We also found a small amount of oxidative form of 4 as a side product (Figure 1B). After HPLC purification, 4 could be obtained in good purity, and the yield was estimated to be more than 3 mg/L. The following process was the installation of an auxiliary at the K48 side chain of protein 4. To this end, all of the free amines were protected by a Boc group. Subsequently, the removal of the Proc group was conducted using Pd catalyst.9 The reaction was monitored by HPLC, and the deprotection process proceeded efficiently to completion within 1 h. Then the auxiliary was introduced directly into the side chain amino groups of the above product by condensation reaction. Finally, Boc group was efficiently removed to release free Lys′ in 20 min by using the reagent TFA/TIPS/H2O (95/2.5/2.5). After ether precipitation and purification, homogeneous protein 5′ was obtained in an overall 45% yield. Followed by further removal of the thiazolidine protection group by methoxylamine, we eventually obtained the key building block 5 (36% overall yield) (Figure 1B). HPLC and ESI-MS analysis confirmed the purity and correctness of final product (Figure 1B,C). We would emphasize that the high removal efficiency of the Proc and Boc pair protection group greatly contributes to efficiently acquiring of the auxiliary modified Ub isomer, despite the protection− deprotection steps. It also worth noting that the Boc/Alloc

Figure 1. (A) Synthetic route toward auxiliary modified Ub isomer 5. Reagents and conditions: (i) Mesna, NH2NH2·HCl, TCEP; (ii) (1) Boc2O, DIEA, DMSO, (2) Pd(PPh3)4, PhSiH3, DMSO, (3) 2,5dioxocyclopentyl 2-(4-(2,4-dimethoxyphenyl)thiazolidin-3-yl)acetate, DIEA, DMSO, (4) TFA/TIS/H2O (95/2.5/2.5); (iii) 6 M Gn·HCl, 0.2 M phosphate, 0.4 M MeONH2, pH 4.0. The more detailed reaction condition was described in SI. (B) HPLC traces (214 nm) for the synthesis of 5 (oxidative form of 4 is labeled with an asterisk). (C) The ESI-MS of final product 5 (obsd 8774.8 Da, calcd 8774.9 Da). Pyl G76C) by using pyrrolysyl-tRNA synthetase (PylRS)-tRNA CUA pair. The reasons for choosing Proc-Lys are that (1) Proc-Lys can be effectively embedded into the expressed protein,9 (2) Proc has been used as an amino protecting-group recently and can be effectively removed by Pd catalyst,9,12 and (3) that the complete orthogonality of the Proc and tert-butoxycarbonyl (Boc) groups allows their independent removal.13 Recombinant

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DOI: 10.1021/acs.orglett.7b03515 Org. Lett. XXXX, XXX, XXX−XXX

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to obtain protein 7 in 75% isolated yield. Then, the ligation between 7 and 9 were initially conducted at a ratio of 1.3:1 to produce 10. However, we found more hydrolyzed byproducts derived from thioester intermediate 7′, accompanying with only a small quantity of ligation product 10. To solve the problem, we raised the ratio of 7 and 9 to 2.5:1. HPLC analysis showed that although the thioester hydrolysis byproduct was still produced, the ligation was finished within 4 h to yield 10 (10.5% isolated yield, Figure S9). The final product 11 was obtained through the removal of auxiliary. After HPLC purification, we eventually obtained protein 11 on a multimilligram scale (Figure 2B). The correctness and the purity of the K48-Ub3 were identified by ESIMS and SDS-PAGE, respectively (Figure 2B,C). Note that one limitation of this approach is the low efficiency of the N-benzyltype auxiliary, especially when ligation proceeded between two larger protein segments (e.g., diUb and Ub). This problem may be solved by use of a more efficient auxiliary such as a 2mercapto-2-phenethyl group.15 To fold K48-Ub3, the urea-gradient dialysis and size-exclusion chromatography purification (SEC) were carried out for synthetic protein 11 (Figure S6).6a The circular dichroism (CD) spectrum of the resulting protein was also obtained (Figure 2D), which exhibited characteristic absorptions at 208 and 226 nm similar to that of monomeric Ub. To demonstrate the biochemical activity of the chemically synthesized K48-Ub3 11, we performed deubiquitinase (DUB) assays using the K48specific ovarian tumor (OTU) family DUB OTUB1 (Figure 2E).16 Upon treatment of OTUB1, we detect that K48-Ub3 was rapidly hydrolyzed to diUb and mono-Ub within 20 min and completely hydrolyzed to monoub within 1 h. These results indicated that the K48-Ub3 was cleaved by OTUB1 at the K48 linkage to generate Ub monomer. The Ub isomer-based approach was readily extended to the synthesis of atypical polyUb chains. Taking K6-Ub3 14 (Figure 3A) as an example, K6-linked Ub isomer was prepared in a

orthogonal strategy reported previously was also used for the construction of 5.7 Unfortunately, after numerous trials, only small amounts of target products were detected in the crude Alloc deprotection product. To test the ligation efficiency of Ub isomer 5, K48-Ub3 was selected as the first synthetic target. Here, we devised a modular, Ub isomer-based strategy for the synthesis of K48-Ub3 that required the use of only three recombinant Ub segments, i.e., distal-Ub 2, auxiliary modified Ub isomer 5, and proximal-Ub 9. Protein 2 was also obtained using the N−S acyl transfer strategy on Ub(1-G76C) protein 1 to convert its C-terminal Cys to a hydrazide. The preparation of Ub segment 9 was quite similar to that of the synthetic route of 5. A minor difference is that ProcLys was genetically incorporated into residue 48 of a wild type (WT) Ub instead of Ub(1-G76C). Author: Next, auxiliary installation and Thz removal gave protein 9 with 34.9% yield.10 With Ub segments 2, 5, and 9 in hands, we carried out hydrazide-based auxiliary-mediated NCL to assemble K48-Ub3 (Figure 2A). The synthesis started with the

Figure 2. Chemical synthesis of K48-Ub3 11. (A) Synthetic route toward K48-Ub3 11 using the K48-Ub 5. The detailed reaction conditions are described in the SI. (B) HPLC traces (214 nm) and ESI-MS for purified K48-Ub3 11. (C) SDS-PAGE analysis of Ub, Ub2, and Ub3. (D) Circular dichroism spectra of Ub, Ub2, and Ub3 11. (E) Deubiquitination assay of K48-Ub311 by K48-specific OTUB1.

Figure 3. Chemical synthesis of 14 and 15. (A) Schematics of K6linked Ub isomer for the synthesis of 14 and 15. (B) SDS-PAGE analysis of 14 and 15. (C) Circular dichroism spectra of 14 and 15. (D) Deubiquitination of K6,48-Ub3 15 by K48-specific OTUB1.

ligation between 5 and Ub thioester Ub(1−75)-MPAA at a ratio of 1:1.3 to afford diUb 6. The ligation process was almost completed in 4 h, with a small amount of hydrolysis product of protein 2 thioester intermediate. The byproduct might be ascribed to the large steric hindrance of secondary amine in auxiliary as previously described.6a,15 After HPLC purification, we could obtain approximately 10 mg diUb 6 in a single ligation step with an isolated yield of 34%. To remove auxiliary, 6 was treated with a cocktail containing TFA/TIS/H2O (95/2.5/2.5)

similar way as building block 5. We first acquired Ub(1-G76C)/ K6-Proc-lys (2.5 mg/L) through genetic incorporation of ProcLys at K6 site of Ub(1-G76C) followed by the use of the N−S acyl transfer strategy in combination with the Proc/Boc orthogonal protection method to obtain product 12 Ub(1− 75)-NHNH2/K6Aux (1.5 mg/L). Finally, product 14 can be C

DOI: 10.1021/acs.orglett.7b03515 Org. Lett. XXXX, XXX, XXX−XXX

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produced in 1.8% isolated yield through two rounds of sequential hydrazide-based auxiliary-mediated NCL between Ub isomer 2, 12, and 13. To further demonstrate the versatility of our approach, we assembled a mixed-linkage tri-Ub 15 composed of K6- and K48-linkages for the first time (Figure 3A). K6,48-mixed polyUb chain has recently been identified to be assembled by the bacterial effector E3 ligase NIeL.17 Our method presented here offers opportunities to obtain product 15 also through sequential auxiliary-mediated NCL between Ub isomer 2, 12, and 9 (overall isolation yield 2.1%). Purified K6Ub3 14 and K6,48-mixed Ub3 15 were characterized using SDSPAGE (Figure 3B), HPLC, and ESI-MS (Figure S11). The MS/ MS sequencing data verified the existence of a Lys6- and K48linked isopeptide bond (Figure S12). After folding, the CD spectrum (Figure 3C) showed the natural conformation of synthetic product 14 and 15. DUB assays were further performed similar as aforementioned (Figure 3D). The results indicate that the mixed Ub3 was cleaved by OTUB1 at the K48 linkage to generate a shorter K6linked chain. Interestingly, the hydrolysis of K48 linkage K6,48mixed Ub3 was slightly slower when compared with that of homotypic K48-Ub3 under the same conditions, indicating that the access of OTUB1 to K48 isopeptide in the mixed architecture was to some extent hindered by the K6-linked ubiquitin. We also found that some mixed substrates were still present after reactions proceeded for 4 h. In conclusion, we have developed a practical method to prepare polyUb chains by using a premade auxiliary modified Ub isomer as the key building block. An important advantage of this approach is that all Ub monomers can be easily obtained through bacterially recombinant expression. In addition, the use of an orthogonal pair of Proc and Boc groups makes the removal of the protecting groups more moderate and efficient. Moreover, this method takes advantages of auxiliary-mediated native chemical ligation so that the ligation process does not require the use of protection strategy. Using this Ub isomer strategy, chemical synthesis of Lys48-linked tri-Ub and atypical Lys6linked tri-Ub, even a mixed-linkage Lys6, Lys48-linked triUb could be readily realized with practical efficiency on a multimilligram scale. We anticipate that with this method in hand, it is now possible to generate essentially homogeneously linked and mixed linkages for polyUb-related biochemical and biophysical studies.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 91753205, 21572214) and the Fundamental Research Funds for the Central Universities (PA2017GDQT0021).

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03515.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jing Shi: 0000-0003-0180-4265 Yi-Ming Li: 0000-0001-6716-8199 Author Contributions ∥

L.X. and J.-Feng Huang contributed equally. D

DOI: 10.1021/acs.orglett.7b03515 Org. Lett. XXXX, XXX, XXX−XXX