Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones

Article abstract of DOI:10.1021/jacs.8b13213

Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation, followed by native chemical ligation assisted by Seitz's auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from recombinant proteins at about 1.5-6 mg/L expression. The thioether-containing isopeptide bonds in the products are chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BK_C34Ub indicated that the obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for biochemical and biophysical studies.

Full text of DOI:10.1021/jacs.8b13213

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of
Recombinant Histones
Guo-Chao Chu,†,‡,§,# Man Pan,†,# Jiabin Li,∥,# Sanling Liu,∥,# Chong Zuo,†,‡ Ze-Bin Tong,†,‡
Jing-Si Bai,‡ Qingyue Gong,∥ Huasong Ai,† Jian Fan,§ Xianbin Meng,⊥ Yi-Chao Huang,† Jing Shi,§
Haiteng Deng,⊥ Changlin Tian,*,∥ Yi-Ming Li,*,‡ and Lei Liu*,†
†Tsinghua-Peking Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and
Chemical Biology, Center for Synthetic and Systems Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen),
Department of Chemistry, Tsinghua University, Beijing 100084, China
‡School of Food and Biological Engineering, Key Laboratory of Metabolism and Regulation for Major Diseases of Anhui Higher
Education Institutes, Hefei University of Technology, Hefei 230009, China
∥
§Department of Chemistry and School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
⊥MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China
S
* Supporting Information
ABSTRACT: Histone ubiquitination affects the structure and function of nucleosomes through tightly regulated dynamic
reversible processes. The efficient preparation of ubiquitinated histones and their analogs is important for biochemical and
biophysical studies on histone ubiquitination. Here, we report the CAACU (cysteine-aminoethylation assisted chemical
ubiquitination) strategy for the efficient synthesis of ubiquitinated histone analogs. The key step in the CAACU strategy is the
installation of an N-alkylated 2-bromoethylamine derivative into a recombinant histone through cysteine aminoethylation,
followed by native chemical ligation assisted by Seitz’s auxiliary to produce mono- and diubiquitin (Ub) and small ubiquitin-like
modifier (SUMO) modified histone analogs. This approach enables the rapid production of modified histones from
recombinant proteins at about 1.5−6 mg/L expression. The thioether-containing isopeptide bonds in the products are
chemically stable and bear only one atomic substitution in the structure, compared to their native counterparts. The
ubiquitinated histone analogs prepared by CAACU can be readily reconstituted into nucleosomes and selectively recognized by
relevant interacting proteins. The thioether-containing isopeptide bonds can also be recognized and hydrolyzed by
deubiquitinases (DUBs). Cryo-electron microscopy (cryo-EM) of the nucleosome containing H2BKC34Ub indicated that the
obtained CAACU histones were of good quality for structural studies. Collectively, this work exemplifies the utility of the
CAACU strategy for the simple and efficient production of homogeneous ubiquitinated and SUMOylated histones for
biochemical and biophysical studies.
enzymes.4−6 It has been shown that ubiquitination or
SUMOylation can affect the stability of the nucleosome and
INTRODUCTION
■
Histone post-translational modifications (PTM) such as
regulate transcriptional activation, DNA replication, and DNA-
damage repair.7−9 Such regulation is achieved through the
recognition or processing of ubiquitinated or SUMOylated
acetylation, methylation, and ubiquitination are essential in
the dynamic regulation of chromatin, which has been described
in organisms ranging from yeast to humans.1−3 Two types of
protein modifiers, Ub and SUMO, can be installed onto the
specific Lys sites of histones to form isopeptide bonds under
ATP-dependent activation by a cascade of E1/E2/E3
Received: December 10, 2018
© XXXX American Chemical Society
A
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nucleosomes by different interacting proteins. For example, a
nucleosome containing both Lys15-ubiquitinated histone H2A
(H2AK15Ub) and Lys20-dimethylated histone H4
(H4K20me2) can recruit p53-binding protein (53BP1) during
the process of DNA damage repair, thereby triggering the
downstream repair process.10 Insights into the structure and
functional regulation of nucleosomes can be inferred by the
elucidation of the molecular mechanisms by which ubiquiti-
nated or SUMOylated histones interact with proteins,11,12 but
such studies are predicated upon the availability of reasonable
amounts of site-specifically ubiquitinated or SUMOylated
histones.7,11
Scheme 1. Combination of Cys Aminoethylation with
Auxiliary-Mediated NCL
a
The enzymatic synthesis of ubiquitinated or SUMOylated
histones remains difficult because the enzyme responsible for a
given modified histone may be unknown or difficult to isolate
in an active form.9,13 Total chemical protein synthesis14,15 can
also be used to access ubiquitinated and SUMOylated
histones16−21 but is technically too difficult for the majority
of biochemistry laboratories to carry out. Meanwhile, although
semiprotein synthesis from expressed histone segments avoids
lengthy peptide synthesis, this technology is often restricted to
ubiquitination and SUMOylation close to N or C termi-
ni.9,22−25 For these reasons, strategies that take advantage of
the unique reactivity of the nucleophilic Cys sulfhydryl
group26−31 are more often used for the site-specific
conjugation of Ub to recombinant full-length histones. These
strategies are mostly based on the construction of isopeptide
bond analogs at the selected Cys through a disulfide-,
hydrazide-, or 1,3-dichloroacetone (DCA)-derived link-
age.13,30−34 In terms of application, Wolberger and Brik et al.
prepared a DCA-derived H2BK120Ub analog and incorpo-
rated it into NCPs (nucleosome core particle) to generate a
crystal structure with the Ubp8/Sgf11/Sus1/Sgf73 DUB
module.35 Muir et al. prepared a disulfide-based
H2BK120Ub-containing nucleosome bearing site-specifically
installed photo-cross-linkers and used it to study the activation
mechanism of histone H3 methyltransferase hDot1L.33
Despite these advances, the labile nature of the disulfide limits
its use under reducing conditions, and DCA or hydrazide
mimics may not adequately imitate the native isopeptide
bond.13,36 Therefore, we wished to examine whether there is
an alternative Cys-based method for the synthesis of
ubiquitinated or SUMOylated histone analogs based upon a
more stable and more structurally similar isopeptide bond
mimic.
Here, we report a cysteine-aminoethylation-assisted chem-
ical ubiquitination (CAACU) strategy for making ubiquiti-
nated or SUMOylated histone analogs from recombinant
proteins. The key step of CAACU is the site-specific
installation of an N-alkylated 2-bromoethylamine derivative
onto the Cys residue of recombinant histones (Scheme 1a).
Ubiquitination and even SUMOylation can then be accom-
plished through auxiliary (Aux)-mediated native chemical
ligation (NCL) with recombinant Ub or SUMO hydrazides.
Because both histones and Ub (or SUMO) are readily
acquired through recombinant expression, the use of
CAACU enables multimilligram quantities of homogeneous
mono- and di-Ub, and SUMO-modified histone analogs are
rapidly obtained. The resulting ubiquitinated or SUMOylated
histone analogs are chemically stable and differ from the native
isopeptide bond by only one atom, and their utility for
biochemical and biophysical studies on histone and nucleo-
some ubiquitination and SUMOylation is established herein.
a
(a) Retrosynthetic analysis of acyl-thialysine formation. The key
reagent in the strategy is the pre-designed bifunctional handle that
bears two reactive units: an N-alkylated 2-bromoethyl amine scaffold,
along with an auxiliary group allowing the NCL. (b) Synthesis of 3
through three steps in 18% overall yield.
RESULTS AND DISCUSSION
■
Cys Aminoethylation with a Bifunctional Handle. The
Cys aminoethylation reaction was originally used by Lindley in
1956 to convert a Cys residue in a protein to a Lys-mimicking
structure.37 In 2007, Shokat et al. reported an important and
hitherto widely used application of this chemistry for the
production of methylated histone analogs.38 Nonetheless, Cys
aminoethylation cannot be directly used to acquire proteins
bearing acyl-Lys-type modifications (Scheme S1a). Here, we
envisaged that a 2-bromoethylamine derivative carrying a
removable NCL-auxiliary would enable the generation of the
acyl aminoethylcysteine structure (Scheme 1a). However, our
initial attempts to synthesize a 2-bromoethylamine derivate
containing the commonly used acid-cleavable N-benzyl-type
auxiliary39 failed because β-bromination of an amine bearing an
acid-sensitive benzyl group proved very difficult (Schemes
S1b).
Fortunately, Seitz et al. recently invented a new type of
removable auxiliary bearing a 2-mercapto-2-phenethyl group.40
We hypothesized that the absence of α-branching at the central
amino group of this auxiliary might allow the required β-
bromination under acidic conditions. With this in mind, we
converted styrene to compound 1 in 95% yield via the AIBN
(2,2′-azobis(isobutyronitrile))-promoted addition of 3-bromo-
2-oxazolidinone. AcmSH was then used to displace the
bromine in 1 to produce compound 2 in 68% yield. Finally,
the ring opening of the oxazolidinone moiety of 2 was carried
out in HOAc containing 31% HBr41 to afford the desired 2-
bromoethylamine derivative in 27% yield (Scheme 1b). Thus,
key bifunctional handle 3 can be rapidly prepared in three
steps in an overall yield of 18%, which bodes well for its future
application.
Next, we used a Cys-containing model peptide to examine
the aminoethylation of 3 under different conditions (Figures
S5 and S6). At pH 8.0−8.5, the aminoethylation reaction
proceeded at high rates in HEPES buffer, converting the model
peptide to the desired modification product in yields of 85−
90% (by HPLC) within 5 h. We then examined the efficiency
of the aminoethylation reaction at different concentrations of 3
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from 10 to 60 mM. It was found that although the reaction was
faster at 40−60 mM 3, bis-alkylated side products were
observed under these conditions (Figure S6). Thus, to
generate the desired product in good homogeneity, the
concentration of 3 was maintained at 20−30 mM.
Ub at K34, a PTM recently found to play an important role in
regulating gene transcription, was examined first.43,44 The
synthesis route of H2BKC34Ub is shown in Figure 2a. A key
substrate required for the synthesis was Ub(1-76)-Mesna or its
hydrazinolysis product Ub(1-76)-NHNH2 (11), which could
be produced in 4 to 5 mg/L of LB medium through intein
technology.45 To improve the efficiency of the synthesis of 11,
we devised a new approach wherein the hydrazinolysis of Ub
variant Ub(1-77D) was accomplished using Ub C-terminal
hydrolase YUH1.46 Thus, Ub(1-77D) was expressed (>50 mg/
L LB medium) first and was then treated with aqueous
hydrazine (5% v/v) in the presence of YUH1 (1 μM) at pH
7.4 and 0 °C. This led to the formation of 11 (80%
conversion) within 2 h in a yield of 30−40 mg from 1 L
expression.
With 11 in hand, we removed the Acm protecting group of 5
using AgOAc or PdCl2 to produce 10.47,48 After purification,
10 was ligated with 11 using a hydrazide-based auxiliary-
assisted NCL49,50 at pH 6.9 to generate 12 within 6 h (44%
isolated yield). Note that the conversion of 10 to 12 was
observed to be almost quantitative by HPLC reaction
monitoring, a surprising result given that use of the common
N-benzyl-type auxiliary often leads to significant hydrolysis
during ligation. This finding exemplifies the advantage of
Seitz’s new auxiliarythe absence of α-branching at the
central amino group.51 Finally, the auxiliary group on 12 was
readily removed within 10 h in 85% conversion (Figure 2c).40
After purification and lyophilization, we obtained H2BKC34Ub
(13) in an isolated yield of 48%. The identity and purity of 13
were ascertained by HPLC, ESI-MS, and SDS-PAGE analysis
(Figure 2b,h). MS-MS analysis further confirmed that Ub was
installed at the correct site (Figure 2d).
The optimized conditions were then applied to full-length
histones. Because only one Cys is present in the four core
histones (Cys110 in H3), histones are particularly amenable to
Cys-based conjugation.13,31 We first tested the Cys amino-
ethylation of 3 with the histone H2B mutant containing a Cys
at position 34 (H2BK34C, 4). To obtain 4, the gene of
Xenopus laevis histone H2B containing the K34C mutation was
cloned into the pET15 vector and expressed in E. coli.42 After
purification from the inclusion body, H2BK34C was obtained
in a 50−60 mg/L LB medium. For the reaction between 3 and
H2BK34C, aqueous buffer containing 6 M guanidine hydro-
chloride (Gn·HCl) was used. Tris(2-carboxyethyl) phosphine
(TCEP) and free methionine were added to maintain the
reducing conditions and avoid oxidation of the thioether bond.
The reaction was performed at pH 8.5 with 20 mM 3 for 5 h.
HPLC analysis showed that the conversion of H2BK34C was
50%, even after the reaction was extended to 10 h. A key
finding was that the addition of a second batch of 3 (20 mM)
and TCEP to the reaction mixture after 5 h could greatly
improve conversion, and ca. 85% of desired product 5 was
obtained in a total of ca. 9 h (Figure 1a). The same protocol
The above results demonstrate the effectiveness of CAACU
for the synthesis of ubiquitinated histones in just four steps:
Cys aminoethylation, Acm deprotection, NCL, and auxiliary
removal. In a typical experiment starting with the expression of
H2BK34C from 1 L of LB medium, we could generate ca. 6
mg of H2BKC34Ub in an overall yield of 12% in ca. 2 weeks
(including reaction, separation, and lyophilization). Using the
same strategy, we could readily conjugate Ub onto the
designed sites of other core histones. For instance, we
successfully synthesized H2AKC119Ub (1.5−2 mg from 1 L
expression of H2AK119C), which was reported to mediate
DNA-damage-induced local transcriptional repression.52 We
also obtained H2AKC13Ub and H2AKC15Ub (3 to 4 mg from
1 L expression of H2AK13C or H2AK15C), which correspond
to the ubiquitination at the N-terminal tail of histone H2A and
play important roles in DNA-damage repair.53 Moreover, we
tested CAACU for the SUMOylation of histone H4, a
modification that was recently reported to be involved in the
modulation of chromatin compaction and the methylation
state.54 SUMO3-intein fusion protein was expressed with the
Cys47Ser mutation. Treatment of this intein fusion protein
with aqueous hydrazine (5% v/v) led to the formation of
SUMO3-(2-92)-C47S-NHNH2 (12−15 mg/L of LB medium,
Figure S9d). This SUMO hydrazide could be readily ligated
with H4 modified with an auxiliary at position 12, leading to
the formation of H4KC12SUMO3 (2 mg from 1 L expression
of H4K12C). This SUMOylated histone was successfully
characterized by HPLC, high-resolution ESI-MS, and SDS-
PAGE (Figure 2e,h).
Figure 1. Incorporation of 3 into histones. (a) Installing 3 into
H2BK34C under the following reaction conditions: protein (10 mg/
mL), 6 M Gn-HCl, 0.5 M HEPES, 10 mM TCEP, 20 mM 3, 37 °C,
pH 8.5. After 5 h, additional 20 mM 3 and 10 mM TCEP were
supplied, and ESI-TOF MS monitoring showed that the conversion of
H2BK34C exceeded 80% after 9 h. (b) ESI-TOF MS showed that 3
could be cleanly installed in different histones. Starting materials are
labeled “S”, and species corresponding to the correct product mass are
labeled “P”.
was also tested for other histones (H2AK13C, H2AK15C,
H3K56C, and H4K12C) to form 6, 7, 8, and 9. To our delight,
all reactions proceeded smoothly with conversions ranging
from 60 to 85% (Figure 1b).
Synthesis of Ubiquitinated and SUMOylated Histone
Analogs. After validating the efficient Cys aminoethylation
reaction of 3, we turned to the synthesis of the ubiquitinated
histone analogs through auxiliary-mediated NCL. H2B bearing
Finally, we wished to show that the CAACU strategy could
also be used to make oligo-Ub analogs and histones modified
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Figure 2. Semisynthesis of Ub- and SUMO-modified histone analogs through the CAACU strategy. (a) The synthesis route of H2BKC34Ub.
Reaction conditions: (i) 5 (0.5 mM, 1.0 equiv), AgOAc (15 mM, 30 equiv), H2O/CH3COOH = 1:1 (v/v), 37 °C, 12 h. (ii) Ub(1-77D) (15−20
mg/mL), YUH1 (1.0 μM), 50 mM Tris buffer containing 5% (v/v) NH2NH2, pH 7.4, 0 °C, 1 h. (iii) 10 (0.5 mM, 1.0 equiv) was ligated with 11
(0.75 mM, 1.5 equiv) through hydrazide-based NCL. (iv) Removing the auxiliary group of 12 (2 to 3 mg/mL) was carried out in denatured buffer
containing 6.0 M Gn-HCl, TCEP (400 mM), morpholine (2.0 M), pH 8.5, 37 °C, 12 h. (b) Analytical RP-HPLC (214 nm) and high-resolution
ESI-MS (average isotope) characterizations of purified 13. (c) HPLC traces (214 nm) for the synthesis of H2BKC34Ub. The NCL between 10 and
11′ (Ub(1-76)-MPAA thioester) was monitored at 0, 1.5, 3.0, and 6.0 h, respectively. 10′ and 11′′ indicate other byproducts and Ub(1-76)-
COOH, respectively. (d) MS-MS analysis of H2BKC34Ub demonstrated the installation of the Ub unit at the correct position. (e) Analytical RP-
HPLC (214 nm) and deconvoluted mass of purified H4KC12SUMO3, H2AKC13/15Ub, and H2AKC119Ub. (f) Synthesis route of Lys27-linked
diubiquitinated H2A (H2AKC13/15-KC27diUb, 19, 20). (g) Analytical RP-HPLC (214 nm) and deconvoluted mass of purified 19 and 20. (h)
SDS-PAGE analysis of the semisynthetic Ub- and SUMO-modified histone analogs.
with oligo-Ubs. To give an example, we chose H2A carrying
Lys27-linked di-Ub on Lys13 or 15, which was previously
reported to play roles in 53BP1-mediated DNA damage repair
(Figure 2f).55,56 First, UbK27C-NHNH2 (14) was reacted
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smoothly with 3 through Cys aminoethylation to generate 15
(ca. 85% HPLC conversion, Figure S10b). After the removal of
Acm from 15, product 16 was successfully ligated with Ub(1-
76)-NHNH2 (11) through hydrazide-based ligation in 5 h
(isolated yield = 51%, Figure S10c). In this fashion, the desired
di-Ub KC27diUb-NHNH2 (17) was obtained, which was then
ligated with H2A bearing an auxiliary at position 13 or 15.
After the removal of the auxiliary, H2AKC13-KC27diUB (19)
or H2AKC15-KC27diUB (20) was successfully obtained (ca. 3
mg from 1 L expression of H2AK13C or H2AK15C) and fully
characterized by HPLC, ESI-MS, and SDS-PAGE (Figure
2g,h). Two conclusions can be drawn from the above
experiments: First, through the use of K-to-C mutated Ub
hydrazide, different di- and, in principle, oligo-Ub can be
conveniently obtained from recombinant materials. Second,
oligo-Ub hydrazide can also be used in CAACU, leading to
substrate proteins modified with oligo-Ub at a predesignated
position. Studies toward these targets are ongoing in our
laboratory.
Nucleosome Reconstitution and Reader Recognition.
The reconstitution of ubiquitinated or SUMOylated nucleo-
somes enables studies on their interactions with reader
proteins. Here, we examined the in vitro reconstitution of
NCPs with the synthetic ubiquitinated histone analogs from
the CAACU strategy. First, H2BKC34Ub, H2AKC13Ub, or
H2AKC15Ub was individually incorporated into octamers
together with other core histones and purified by size-exclusion
chromatography (Figure S13). Then the octamer was
reconstituted into nucleosomes with a 147 base pair 601
DNA sequence through stepwise dialysis. SDS-PAGE and
native-gel analyses confirmed that synthetic ubiquitinated
histones were stoichiometrically incorporated into the
nucleosomes (Figure 3a,b).42
Figure 3. Nucleosome reconstitution and selective recognition of the
interacting protein. (a) SDS-PAGE analysis of reconstituted histone
octamers containing ubiquitinated H2B and H2A. (b) Sybr Gold-
stained native gels of reconstituted NCPs containing ubiquitinated
H2B and H2A. (c and d) GST-Tudor-UDR pull-down assays of
NCPs containing ubiquitinated H2A analogs and H4KC20me2 in
which H2AK13Ubwt-containing NCP were used as the control [(c)
Coomassie-stained and (d) immunoblotting anti-H2A]. (e) Gel
filtration chromatography of the preformed NCP-UbmeK15/GST-
53BP1 complex. (λ = 280 nm, Superdex 200 Increase 10/300 GL). (f)
SDS-PAGE analysis of the peak fraction of the NCP-ubmeK15/GST-
53BP1 complex, stained with Coomassie blue, in which the elution
volume of each sample was labeled.
Next, we tested the selective recognition properties of the
ubiquitinated nucleosomes with the interacting proteins
(readers). 53BP1 is a critical regulator of the cellular response
to DNA double-strand breaks and was recently discovered to
bind specifically to NCPs containing both H4-K20me2 and
H2AK15Ub but not H2AK13Ub.10,55 Here we used NCPs
containing H2AKC13Ub or H2AKC15Ub to do the test and
performed pull-down assays with GST-tagged 53BP1 consist-
ing of the tandem Tudor-UDR segment (GST-Tudor-UDR,
residues 1484−1631) as previously described.55 H2AKC13Ub
or H2AKC15Ub was incorporated into NCPs together with
H4KC20me2 (made by Shokat’s strategy).38 The resulting
NCPs as well as an NCP containing wild-type H2AK13Ub
(H2AK13Ubwt) as a control were incubated with GST-Tudor-
UDR. After the removal of nonspecific binding proteins by
thorough washing of the glutathione-sepharose resin, the
samples were resuspended directly in protein loading buffer
and then analyzed by SDS-PAGE. It was found that 53BP1
Tudor-UDR protein selectively bound to NCP containing
H2AKC15Ub but not to NCP containing H2AKC13Ub or
H2AK13Ubwt (Figure 3c). The pull-down experiments were
also repeated and analyzed by immunoblotting using an anti-
H2A specific antibody, with the same result (Figure 3d). In
addition, size exclusion chromatography and SDS-PAGE
analysis further confirmed that NCP containing H2AKC15Ub
could form a stable complex with 53BP1 (Figure 3e,f). These
results suggest that NCP containing the ubiquitinated histones
prepared by CAACU exhibit similar molecular recognition
properties to their natural counterparts.
Sulfur-Containing Isopeptide Bonds Can Be Recog-
nized and Hydrolyzed by DUBs. Histone deubiquitination
catalyzed by various DUBs occurs in the processes of DNA
damage repair, gene activation inhibition, and chromosome
condensation,57 and its dysfunction is implicated in many
pathological processes such as cancer, aging, and infertility.58
Site-specifically ubiquitinated histones and nucleosomes are
needed for biochemical and screening studies of histone DUBs,
but analogs bearing 1,3-dichloroacetone moieties are not
hydrolyzable substrates. Previously, Strieter et al. demonstrated
that synthetic diUbs with thioether-based isopeptide bonds are
functionally active substrates for DUBs.59 We wished to
examine whether ubiquitinated histones made by CAACU
were also functionally active substrates for DUBs. To this end,
we treated H2AKC13Ub with two representative DUBs,
namely, YUH1 and UCHL1.60 For direct comparison, native
H2AK13Ub was tested alongside H2AKC13Ub synthesized by
CAACU. SDS-PAGE monitoring showed that H2AK13Ub was
rapidly hydrolyzed by YUH1 to Ub and H2A in 0.5 h.
However, even when the incubation time was extended to 1.5
h, very little H2AK13Ub was hydrolyzed by UCHL1. These
observations indicate that the two DUBs had very different
reactivity toward H2AK13Ub, an interesting phenomena that
was also observed for H2AKC13Ub (Figure 4a, 4b). We also
found that YUH1 can rapidly hydrolyze H2AKC15Ub while
UCHL1 cannot (Figure S16). Therefore, the thioether-
containing isopeptide bond could be processed by DUBs to
a similar extent as for a native isopeptide bond.
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Figure 4. Ubiquitinated histones made through CAACU are
functionally active substrates for DUBs. (a, b) DUB assays of
YUH1 (2.5 μM) and UCHL1 (2.5 μM) toward H2AK13Ubwt (2.5
μM) and H2AKC13Ub (2.5 μM). (c) DUB assay of YUH1 (5.0 μM)
toward nonhydrolyzable H2AKC15UbAux (2.5 μM). (d) Non-
hydrolyzable H2AKC15UbAux (0, 1.0, 2.5, 5.0, and 7.5 μM) acts as
a competitive inhibitor of Ub-AMC (2.0 μM) cleavage by YUH1 (10
nM). Excitation wavelength = 360 nm and emission wavelength = 460
nm.
Figure 5. H2AKC13Ub photoaffinity probe. (a) Synthesis of probe
22. The aryl azide photo-cross-linker was installed at the position
close to the isopeptide bond. (b) Analytical RP-HPLC (λ = 214 nm),
ESI-MS, and deconvoluted mass of purified H2AKC13Ub probe. (c)
SDS-PAGE and Western blot analysis (anti-H2A antibody) of the
photo-cross-linking reaction between DUBs (YUH1 or UCHL1, 5.0
μM) and the H2AKC13Ub probe (2.5 μM) in the presence or
absence of ultraviolet light irradiation (λ = 365 nm). * indicates the
self-cross-linked product of the probe.
Meanwhile, we tested the hydrolytic activity of YUH1 on
H2AKC15Ub that still carried the auxiliary group at its
isopeptide bond (H2AKC15UbAux). H2AKC15UbAux was found
to be almost impervious to hydrolysis by YUH1 (Figure 4c),
consistent with the previous finding that the N-alkylated
isopeptide bond is resistant to DUBs.61 Nonetheless, our
fluorescence assay showed that H2AKC15UbAux was a
competitive inhibitor of YUH1 during its cleavage of Ub-
AMC (Ub-7-amino-4-methylcoumarin). This finding suggests
that binding takes place between H2AKC15UbAux and YUH1
(Figure 4d) and that the development of profiling tools based
on photo-cross-linking might be possible.62,63
To test this idea, we introduced an aryl azide photo-cross-
linker at the auxiliary of H2AKC13Ub to construct a
photoaffinity probe (22, Figure 5a,b). Probe 22 was incubated
with YUH1 and UCHL1 at 0 °C for 1 h and then exposed to
365 nm UV light for 5 min. An analysis of the reaction mixture
by SDS-PAGE and immunoblotting revealed the expected
cross-linked band of probe 22 and YUH1 at the correct
molecular weight position. This band was attributed to
covalent cross-linking because no band was detected at this
position in the absence of UV irradiation. In contrast, 22 did
not cross-link with UCHL1 (Figure 5c), consistent with the
aforementioned observation that UCHL1 was not active on
H2AK13Ub. Collectively, our experiments suggest that the
thioether-containing isopeptides in ubiquitinated histones
made through CAACU were hydrolyzable by DUBs. Thus,
the histone analogs made by CAACU may constitute
functionally active reagents for the profiling and study of
DUBs at the histone or nucleosome levels.
to enable facile access to multimilligram quantities of
ubiquitinated or SUMOylated histone analogs. We sought to
ascertain whether these synthetic proteins could also be used
for structural studies on ubiquitinated nucleosomes through
single-particle cryo-EM (cryo-EM).
Accordingly, we examined the nucleosome containing
synthetic H2BKC34Ub (i.e., H2BKC34Ub-NCP) by cryo-EM.
H2BKC34Ub-NCP was reconstituted, and purified samples of
it were flash frozen. Single-particle cryo-EM pictures were
recorded using a Tecnai F20 electron microscope operated at
200 kV equipped with a Falcon-3 direct electron detector.
After 2D/3D classification and refinement, we obtained three
different types (types I−III) of H2BKC34Ub-NCP cryo-EM
structures at average resolutions of 7.1, 10.0, and 11.7 Å,
respectively (Figure S18c and Figure S20). Surface views of
these three structures show them to be disklike and consistent
with classic NCP structures64,65 and bear extra density
attributable to Ub on the side of NCP. The density attributed
to Ub is significantly different among the three structures,
consistent with a previous theory that the Ub domains in
ubiquitinated nucleosomes are structurally mobile.10,35,66
In the type-I structure, the DNA duplex and core secondary
elements of the histone octamers fit unambiguously onto the
cyro-EM map, with only one protrusion corresponding to the
Ub linked to the upper K34 residue (Figure 6a and Figure
S19). The density for the Ub moiety was lower than for the
rest of the structure, and the local resolution of the protruding
part was lower than that of NCP (Figure S18e). Nevertheless,
we could locate the protruding density adjacent to the K34 of
H2B. Furthermore, the DNA densities adjacent to the K34 of
H2B in the cryo-EM map deviated from those in the native
NCP structure, causing an expansion of the distance between
the two DNA duplexes within H2BKC34Ub-NCP by ca. 2 Å
(Figure 6b). The resulting space was large enough to allow the
Cryo-EM Studies on a Ubiquitinated Nucleosome
Containing H2BKC34Ub. Structural studies of ubiquitinated
nucleosomes and their complexes with many interacting
proteins have yielded fundamental insight into the recognitory
and regulatory mechanisms of chromatin ubiquitination.10,35
An essential prerequisite for such studies is the ready
availability of high-quality, homogeneous ubiquitinated NCP.
The above results exemplify the utility of the CAACU strategy
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Figure 6. Cryo-EM structure of type-I H2BKC34Ub NCP. (a) Surface
of type-I H2BKC34Ub NCP viewed along the DNA axis and the
orthogonal direction. Rigid-body fitting of the nucleosome crystal
structure (DNA, PDB code 1KX5; octameric histones, PDB code
2CV5) into H2BKC34Ub NCP density is shown as ribbons (purple,
DNA; yellow, histone H2BK34C; red, K34 of H2B). Ub (green
color) could not be readily placed in the attributed density. (b) DNA
densities in the map deviate from those in the nucleosome crystal
structure.
Ub modifier to pass through the DNA duplexes. Secondary
elements of the type II and III structures could not be assigned
because of their poor resolution, but the protruding densities
adjacent to the lower K34 (type II) or upper and lower K34
(type III) were apparent (Figure S21). The Ub domain could
not be readily identified in the attributed density because of
the structural flexibility. Note that the cryo-EM structures of
H2BKC34Ub-NCP solved here were of a higher resolution but
otherwise were almost identical to that of their previously
reported, native counterpart obtained in a laborious total
synthesis.42 We thus anticipate that the ubiquitinated histone
analogs prepared by CAACU would be suitable for structural
studies on nucleosome modifications.
Short-Chain Lys Acylation. Apart from histone ubiquiti-
nation and SUMOylation, a number of interesting short-chain
Lys acylations (e.g., β-hydroxybutyrylation and succinylation)
have been mapped on histones.67 The functional and structural
roles of many of these Lys acylation modifications are
inadequately characterized, in part because of the difficulty in
obtaining site-specifically acylated histones or analogs.
Although methods such as thiol−ene ligation, Staudinger
ligation, and hydrazide mimics have been developed, new and
complementary strategies will improve the capability of the
tool box.30,68,69
To demonstrate that the central concept of CAACU (i.e., a
combination of Cys aminoethylation and protein ligation) is
also suitable for the synthesis of short-chain Lys acylated
histones, we sought to synthesize an analog of H2B bearing
Lys β-hydroxybutyrylation at K34 (i.e., H2BKC34Bhb). This
new type of epigenetic regulatory marker was recently
identified by proteomics and proposed to be involved in
regulating gene expression.70 However, an in vitro preparation
of the full-length histone bearing Lys β-hydroxybutyrylation or
an analog has yet to be reported. In our experiment, we used
the Bhb-thioester, obtained in one step from (R)-3-
hydroxybutyric acid in 58% yield, to ligate with the auxiliary-
containing H2BK34C derivative (10) (Figure 7a). After
removal of the auxiliary, desired product H2BKC34-Bhb was
Figure 7. Site-specific incorporation of short-chain Lys acylation
analogs into histones. (a and d) Strategy for the synthesis of
H2BKC34Bhb and H2BKC34Succ. (b and e) Analytical RP-HPLC
chromatogram (λ = 214 nm) and deconvoluted mass of purified
products. (c and f) Tandem MS analysis of the trypsinized KCBhb-
and KCSucc-containing peptide segments.
obtained in 4 to 5 mg from 1 L expression of H2BK34C
(Figure 7b). MS-MS analysis revealed a peptide segment
containing H2BKC34Bhb, which confirmed β-hydroxyisobutyr-
ylation at the K34 residue (Figure 7c).
In addition, we synthesized the succinylated H2B
(H2BKC34Succ) analog using the same concept. To avoid
intramolecular cyclization, the carboxylate was protected with a
tert-butyl protecting group (Figure 7d). The desired product
was fully characterized by HPLC, ESI-MS, and MS-MS (Figure
7e,f). Thus, the CAACU strategy also amounts to a general
method of installing short-chain Lys acylation onto histones.
SUMMARY
■
Biochemical analyses of the effects of ubiquitination and
SUMOylation on the structures and functions of nucleosomes
and their complexes are necessary to understand the regulation
of Ub and SUMO modification on transcriptional activation,
DNA replication, and DNA-damage repair. To more easily and
cheaply obtain the requisite modified protein reagents, we
developed the CAACU strategy that combines the powers of
Cys aminoethylation and protein ligation and established it as
an easy-to-implement method for obtaining multimilligram
quantities of ubiquitinated and SUMOylated histones from
recombinantly expressed proteins. The products yielded by this
strategy are chemically stable and exhibit only minor structural
and therefore functional perturbation of the isopeptide bond.
By testing the molecular recognition of reader proteins,
hydrolytic processing by deubiquitination enzymes, and cryo-
EM structural determination, we demonstrated that the
ubiquitinated and SUMOylated histones made by CAACU
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are applicable to biochemical and biophysical studies. Finally,
the CAACU strategy was used to synthesize di-Ub modified
histones and histones bearing Lys succinylation and β-
hydroxybutyrylation, further broadening its scope. Accordingly,
we anticipate that the concept of combining Cys amino-
ethylation and protein ligation may enable the development of
more synthetic methodologies applicable to studies of protein
post-translational modifications.
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ASSOCIATED CONTENT
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■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.8b13213.
́
(10) Wilson, M. D.; Benlekbir, S.; Amelie Fradet-Turcotte, A.;
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Experimental details and compound characterization;
general methods; organic synthesis; protein expression
and purification; octamer and nucleosome reconstitution
and purification; NCP pull-down assays; NCP-ubmeKc15
and GST-53BP1 complex formation; preparation of
protein hydrazide; semisynthesis of the Ub- and SUMO-
modified histone analogs, H2AKC13Ub photoaffinity
probe, and H2BKC34Bhb and H2BKC4Succ through the
CAACU strategy; and deubiquitination, inhibition, and
photo-cross-linking assays (PDF)
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Chemical synthesis of phosphorylated histone H2A at Tyr57 reveals
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AUTHOR INFORMATION
Corresponding Authors
*cltian@ustc.edu.cn
*ymli@hfut.edu.cn.
*lliu@mail.tsinghua.edu.cn.
ORCID
■
Jing Shi: 0000-0003-0180-4265
Lei Liu: 0000-0001-6290-8602
(18) Bi, X.; Pasunooti, K. K.; Liu, C. F. Total chemical and
semisynthetic approaches for the preparation of ubiquitinated
proteins and their applications. Sci. China: Chem. 2018, 61, 251.
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sequential protein synthesis: the case of ubiquitinated and
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mediated rapid deprotection of N-terminal cysteine under native
chemical ligation conditions for the efficient preparation of syntheti-
cally challenging proteins. J. Am. Chem. Soc. 2016, 138, 5069.
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synthesis of histone H2A with methylation at Gln10. Sci. China:
Chem. 2017, 60, 621.
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W. Chemically ubiquitylated histone H2B stimulates hDot1L-
mediated intranucleosomal methylation. Nature 2008, 453, 812.
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Author Contributions
#G.C.C., M.P., J.B.L. and S.L.L. contributed equally to this
work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported by the National Key R&D Program
of China (2017YFA0505200 and 2017YFA0505400) and the
NSFC (91753205, 21532004, 21877024, 81621002, 21621003
and 21708036). We thank the protein chemistry facility at the
Center for Biomedical Analysis of Tsinghua University for
sample analysis. We thank the staff at the Center for Integrative
Imaging (University of Science and Technology of China) for
their assistance during cryo-EM data collection.
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