Communication pubs.acs.org/JACS
Genetically Encoded Photoaffinity Histone Marks Xiao Xie,†,# Xiao-Meng Li,§,# Fangfei Qin,‡,⊥,# Jianwei Lin,§ Gong Zhang,‡,⊥ Jingyi Zhao,† Xiucong Bao,§ Rongfeng Zhu,‡,⊥ Haiping Song,† Xiang David Li,*,§ and Peng R. Chen*,†,‡ †
Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China § Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China ‡ Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China ⊥ Academy of Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China S Supporting Information *
Scheme 1. Development of Genetically Encoded Photoaffinity Histone Marksa
ABSTRACT: Posttranslational modifications (PTMs) of lysine are crucial histone marks that regulate diverse biological processes. The functional roles and regulation mechanism of many newly identified lysine PTMs, however, remain yet to be understood. Here we report a photoaffinity crotonyl lysine (Kcr) analogue that can be genetically and site-specifically incorporated into histone proteins. This, in conjunction with the genetically encoded photo-lysine as a “control probe”, enables the capture and identification of enzymatic machinery and/or effector proteins for histone lysine crotonylation.
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ovalent histone modifications, primarily through lysine posttranslational modifications (PTMs), are fundamental epigenetic regulation mechanisms underlying diverse cellular processes. Recently, an array of new lysine PTMs1 have been identified on histones, reflecting the unprecedented diversity and dynamic nature of histone modifications. The functional roles of many of these new lysine PTMs, however, remain unknown.2 A major obstacle lies in the difficulty in discovering the enzymatic machinery (e.g., “writers” and “erasers”) as well as the effector proteins (e.g., “readers”) for these histone marks.2a,3 For example, although the novel histone lysine crotonylation (Kcr) has been found in both the flexible tail and globular domains, and is particularly abundant at the promoter and enhancer regions,4 the molecular details for Kcr-stimulated active transcription remain to be clarified.5 In particular, because the functions of such lysine PTMs are highly context dependent, dissecting the interactions between Kcr and its enzymatic machinery/effector proteins at specific histone sites is highly desired to reveal the distinct biological impact. Photoaffinity amino acids such as diazirine-containing photolysine, photo-leucine, and photo-methionine6 allow the conversion of non-covalent interactions into covalent linkages7a,b that enable specific capture of the weak and transient protein− protein interactions occurring in nature. However, these probes lack the target and site specificity needed for global labeling. In contrast, the genetic code expansion strategy7a,c permits the sitespecific incorporation of photoaffinity amino acids into a protein of interest, offering unique advantages in capturing protein− protein interactions in a site-specific fashion in vitro and in living © 2017 American Chemical Society
(a) “Dual-functional” Pyl analogues bearing a diazirine photoaffinity group and a given lysine PTM (e.g., photoaffinity crotonyl lysine, K*cr) can be genetically and site-specifically incorporated into histone proteins to allow covalent capture of the corresponding enzymes or effector proteins (e.g., “readers” and “erasers”) for this histone mark. (b) The caged photo-lysine analogue bearing a diazirine photoaffinity group and a caging moiety on ε-amine (photoaffinity caged lysine, PNBK*) can be genetically and site-specifically incorporated into histone proteins. Removal of this caging group by reductive enzyme NTR can in situ generate the decaged photo-lysine (K*) with the εamine unmasked. a
cells. This strategy also enables site-specific incorporation of various lysine PTMs into target proteins,8 allowing the welldefined study of these modifications at a desired lysine residue.8d Nevertheless, there is currently no genetically encoded lysine analogue that contains both the photoaffinity group and the PTM moiety. We envisioned that coupling these “dual functions” into the same lysine side-chain may afford a genetically encoded lysine probe capable of in situ capturing the corresponding enzymes or effector proteins for a given lysine PTM. Herein, we Received: February 10, 2017 Published: May 1, 2017 6522
DOI: 10.1021/jacs.7b01431 J. Am. Chem. Soc. 2017, 139, 6522−6525
Communication
Journal of the American Chemical Society
genesis library to select the appropriate PylRS variant. K*cr was subject to green fluorescent protein (GFP)-based screening against the PylRS library (Table S1). To our delight, we identified a MmPylRS mutant that showed a high efficiency for incorporating K*cr into GFP. This MmPylRS mutant was named as K*cr-RS (Leu309Ala, Cys348Phe, Tyr384Trp) (Figure 1b). The resulting GFP variant bearing the site-specifically incorporated K*cr at residue N149 (GFP-N149K*cr) was further subject to ESI-MS analysis, which verified the fidelity of this incorporation (Figure 1c). We then examined the photoaffinity function of the diazirine group on K*cr using our previously studied protein HdeA as a model.12 The bacterial acid chaperone HdeA interacts with a range of client proteins upon acid stress, which can be readily captured by our previously developed diazirine-containing photo-cross-linkers.12,13 K*cr was incorporated into HdeA at residue V58 within its chaperoning region, and subsequent photo-cross-linking at low pH conditions efficiently captured various client proteins as examined by Western blotting (Figure S2), verifying the photo-cross-linking capability of K*cr. Next, we utilized the K*cr-RS-tRNA pair to incorporate K*cr into the desired site on recombinant H3 protein in E. coli. Because lysine crotonylation has been shown to occur on K56 and K79, which are located on the histone tail close to or within the globular domain, we chose to incorporate K*cr at these two sites for proof-of-concept. Modification of these two lysine residues would affect the stability of histone cores in order to control the binding DNA’s transcriptional activity.4c,14 K*cr was successfully incorporated into these two lysine sites on H3 (Figure 1d), and the resulting H3 variants H3K*56cr and H3K*79cr were purified according to the reported procedure.8a,c ESI-MS analysis on wild-type H3 (WT-H3), H3K*56cr, and H3K*79cr verified the fidelity of K*cr incorporation into H3 at different sites (Figures S3 and S4). We then subjected H3K*79cr to trypsin digestion and high-performance liquid chromatography−tandem MS (HPLC-MS/MS), which further confirmed the site-specific incorporation of K*cr into H3 (Figure 1e). Together, we showed that K*cr can be genetically and sitespecifically incorporated into H3 protein. Because the aforementioned photo-lysine itself can only be metabolically introduced into the entire proteome to replace lysine in a global fashion, we next set out to adopt the genetic code expansion strategy to site-specifically incorporate photolysine into a protein of interest. We designed and synthesized the “caged” version of photo-lysine (PNBK*) and evolved the recognition PylRS variant. The free ε-amine on N-boc-photolysine was reacted with 4-nitrobenzyl chloroformate with Et3N as the base in 1,4-dioxane/water (1/1) to give boc-protected PNBK*. The boc group was removed by treatment with 4 M HCl/THF, and the final product PNBK* was obtained after purification with HPLC (Figure 2a and Scheme S4). Following the similar GFP-based 96-well screening protocol described above, we identified the PylRS variant for efficient PNBK* incorporation, which was named as PNBK*-RS (Leu309Ala, Cys348Ala, Tyr384Tyr) (Figure 2b). ESI-MS analysis on PNBK*-incorporated GFP (GFP-N149-PNBK*) with optimized expression conditions in E. coli showed that the majority of the final GFP product contains PNB-decaged photo-lysine at residue N149 (Figures 2c and S5). Furthermore, PNBK* was site-specifically incorporated into H3 protein at K56 and K79 by PNBK*-RS (Figure 2d). ESI-MS analysis verified that the PNBdecaged protein H3K*79 was successfully produced (Figure S6).
report the genetically encoded photoaffinity Kcr analogue (K*cr) that can site-specifically replace a lysine residue on histone to capture the corresponding partner proteins (Scheme 1a). In addition, we also adopted a “decaging” strategy to genetically encode photo-lysine (K*) into histones, which can be used to investigate the specificity of an effector of interest toward the corresponding lysine PTM as opposed to unmodified lysine. A caged photo-lysine (PNBK*) was first genetically incorporated into histone through genetic code expansion and subsequently decaged by reductive enzymes such as nitroreductase (NTR, Figure S1) in E. coli9 to ultimately generate K* at a defined histone site (Schemes 1b and S2). The pyrrolysine (Pyl)-based genetic code expansion system has emerged as a powerful approach to genetically encode unnatural amino acids into diverse prokaryotic and eukaryotic species, multicellular organisms, and even in animals.6a,10 We started by synthesizing K*cr and obtaining a Pyl tRNA synthetase (PylRS) mutant for efficient recognition. The precursor of K*cr, N-boc-photo-lysine, was synthesized according to the previously reported procedure.5b N-Bocphoto-lysine was then reacted with crotonyl-OSu with Na2CO3 as the base in 1,4-dioxane/water (1/1) to give the boc-protected crotonyl photo-lysine. The boc group was removed by treatment with 4 M HCl/THF, and the final product K*cr was obtained after purification with HPLC (Figure 1a and Scheme S3). Because the active-site pocket of PylRS from Mathanosarcina maize (MmPylRS) has been shown to be highly promiscuous in substrate recognition, we adopted our rationally designed 96-well format PylRS mutant library11 instead of the random muta-
Figure 1. Site-specific incorporation of K*cr into GFP and H3 protein in E. coli. (a) Synthetic route of crotonyl photo-lysine (K*cr). (b) Mutation sites on the identified K*cr-recognition PylRS variant (K*crRS). (c) Site-specific incorporation of K*cr into the GFP model protein in E. coli verified by ESI-MS analysis. Expected MW of GFP-N149K*cr, 27 830 Da; measured MW, 27 830 Da (major peak) and 27 696 Da (minor peak, with the N-terminal methionine cleaved). (d) Incorporation of K*cr into H3K56 and H3K79 as examined by Western blotting with anti-His antibody, as the H3 protein was His-tagged. (e) Verifying the K*cr incorporation into H3K79 in E. coli by HPLC-MS/MS analysis. 6523
DOI: 10.1021/jacs.7b01431 J. Am. Chem. Soc. 2017, 139, 6522−6525
Communication
Journal of the American Chemical Society
Figure 3. Sirt3 is the decrotonylation enzyme of H3K79cr and H3K*79cr proteins. (a) Confirmation of interactions between H3K*79cr and Sirt3 by the photo-cross-linking assay. The cross-linked band of H3K*79cr and Sirt3 can be observed upon UV irradiation, while no cross-linked band exists if the H3K4cr competitor peptide is added. No cross-linked band of H3K*79 and Sirt3 was observed by Western blotting against H3. (b) Decrotonylation enzymatic assay using Sirt3 and Sirt5 on H3K79cr and H3K*79cr proteins analyzed by Western blotting. Pan-crotonylation levels show the de-crotonylation activity of Sirt3 but not Sirt5.
Figure 2. “Decaging” strategy to genetically and site-specifically incorporate photo-lysine (K*) into GFP and H3 protein in E. coli. (a) Synthetic route of PNB-caged photo-lysine (PNBK*). (b) Mutation sites on the identified PNBK*-recognition PylRS variant (PNBK*-RS). (c) Verifying the final PNB-decaged GFP product (GFP-N149K*) expressed in E. coli by ESI-MS analysis. Expected MW of GFP-N149K*, 27 762 Da; measured, 27 765 Da (major peak). The minor peak corresponds to the GFP-N149PNBK* protein (expected MW, 27 941 Da; measured, 27 944 Da). (d) Incorporation of PNBK* into H3K56 and H3K79 as identified by Western blotting. (e) Verifying the generation of H3K*79 in E. coli by HPLC-MS/MS analysis.
interference by the diazirine group on K*cr. Nevertheless, the interference on protein−protein interactions by diazirine is less significant than that of other photoaffinity groups,17 and our results confirmed that Sirt3 but not Sirt5 removed the crotonyl group from Lys79 on both H3K79cr and H3K*79cr (Figure 3b). Together, our photo-cross-linking results demonstrated that sitespecific incorporation of K*cr into proteins offered a powerful tool to capture the protein−protein interactions mediated by this unique lysine PTM. Finally, we directly incorporated K*cr into proteins for photocross-linking in living mammalian cells. GFP was first used as the model protein to verify the efficiency and fidelity for encoding K*cr in HEK293T cells (Figures 4a and S10). We also confirmed
Finally, HPLC-MS/MS was further used to confirm the generation of photo-lysine at site 79 on H3 (Figure 2e). Although H3K79 is known to be crotonylated inside cells, the regulation mechanism and function of H3K79cr remain uninvestigated.15 With H3K*79cr protein in hands, it is feasible to capture the potential “erasers” for H3K79cr by utilizing the photoaffinity K*cr to fix the weak and transient PTM-mediated protein interactions. We incubated H3K*79cr with a known histone H3K4cr decrotonylase, Sirt3.16 Upon UV irradiation, a substantial amount of cross-linked H3K*79cr-Sirt3 was observed by Western blotting against H3 (Figure 3a, lane 3), whereas the control experiment using Kcr-incorporated H3 (H3K79cr) showed no such cross-linked band (Figure 3a, lane 5). Notably, this K*cr-induced cross-linking between H3K*79cr and Sirt3 could be in competition with H3K4cr peptide, indicating that this capture is dependent on the substrate-binding domain of Sirt3 (Figure 3a, lane 4). Western blotting using anti-His antibody also confirmed the capability of H3K*79cr to capture Sirt3. In contrast, Sirt5 was not photo-cross-linked with H3K*79cr (Figure S9), due to its preference toward negatively charged modifications (e.g., succinylation and glutarylation). Notably, our photo-lysine-bearing H3 protein (H3K*79) was not able to photo-cross-link with Sirt3, which served as a “control probe” to further verify the specificity of Sirt3 toward Kcr as opposed to unmodified lysine residue on H3 (Figure 3a, lane 6). Inspired by these results, we also performed the decrotonylation enzymatic assay using Sirt3 and Sirt5 on H3K79cr and H3K*79cr proteins. Sirt3 appeared to work less well as a decrotonylase on H3K*79cr as compared to H3K79cr, which is likely due to the slight
Figure 4. Site-specific incorporation of K*cr into GFP and H3 protein in mammalian cells. (a) Verifying the expression of GFP-Y40K*cr protein by Western blotting against the C-terminal His-tag and Myc-tag. (b) Amber suppression efficiency of K*cr-RS in recognizing K*cr at sites H3K56 and H3K79 in mammalian cells as examined by Western blotting against the C-terminal His-tag.
that K*cr and PNBK* were not randomly incorporated into mammalian proteome (Figures S11−S13). We next incorporated K*cr into H3 protein at residues K56 and K79 in HEK293T cells. The expressed proteins can be readily detected by Western blotting using the anti-His antibody against the Cterminal His-tag on these H3 variants (Figure 4b). We then performed photo-cross-linking in living cells bearing the H3K*79cr protein. To our delight, Western blotting analysis showed a distinct, photo-dependent gel band from other nonspecific backgrounds (Figure S14, compare lane 1 with lane 3). In addition, the presence of nicotinamide (NAM), a 6524
DOI: 10.1021/jacs.7b01431 J. Am. Chem. Soc. 2017, 139, 6522−6525
Journal of the American Chemical Society
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sirtuin inhibitor that blocks the only known cellular decrotonylase, Sirt3, further enhanced the intensity of this photo-crosslinked band (Figure S14, lane 2), suggesting that the captured protein(s) might bind to histone H3 in a K79 crotonylationdependent manner. In summary, we have developed photoaffinity analogues of crotonyl lysine and unmodified lysine that can be site-specifically incorporated into proteins such as histone H3 protein via the genetic code expansion strategy. This allowed the investigation of lysine PTM-mediated protein−protein interactions between fulllength H3 protein and its epigenetic regulatory effectors in vitro and in living cells. For example, we efficiently captured the Kcrmediated specific interaction between H3K*79cr protein and the “eraser” protein Sirt3. We further applied direct photo-crosslinking on K*cr-bearing H3 proteins inside living cells, which, in conjunction with the further MS/MS analysis, may uncover new enzymatic machinery and/or effector proteins for histone lysine crotonylation. Such a “dual-functional” photoaffinity lysine analogue can be extended to accommodate additional lysine PTMs other than crotonylation, which may become a powerful toolkit to uncover diverse lysine PTM-mediated histone− effector interactions. Finally, increasing evidence implies that the unmodified lysine residues on histone proteins represent a unique state that can also mediate the interactions between histone and certain effector proteins with distinct epigenetic outputs.4c,18 Therefore, beyond its utility as a “control probe” demonstrated in this study, our decaging-enabled site-specific incorporation of the photo-lysine probe may allow the identification of unmodified lysine-mediated histone−effector interactions.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b01431. General considerations, supplementary methods, and Schemes S1−S4, Figures S1−S18, and Table S1 (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*
[email protected] *
[email protected] ORCID
Gong Zhang: 0000-0002-6992-176X Xiang David Li: 0000-0002-2797-4134 Peng R. Chen: 0000-0002-0402-7417 Author Contributions #
X.X., X.-M.L., and F.Q. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21521003 and 21432002 to P.R.C.; 21572191 to X.D.L). P.R.C. also acknowledges the support from National Key Research and Development Program of China (2016YFA0501500). X.D.L acknowledges the support from the Hong Kong Research Grants Council Collaborative Research Fund (CRF C7029-15G), Areas of Excellence Scheme (AoE/P705/16), General Research Fund (GRF 17303114), and Early Career Scheme (ECS) (HKU 709813P). 6525
DOI: 10.1021/jacs.7b01431 J. Am. Chem. Soc. 2017, 139, 6522−6525
