Hydrazide Mimics for Protein Lysine Acylation to Assess Nucleosome Dynamics and Deubiquitinase Action

Article abstract of DOI:10.1021/jacs.8b03572

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins. We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys, and ubiquityl-Lys that could be recognized selectively by relevant acyl-Lys modification antibodies. The acyl-Lys modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a function of modification type and site. We also installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study the SAGA deubiquitinating module's molecular recognition. These results suggest that acyl-Lys mimics offer a relatively simple and promising strategy to study the role of acyl-Lys modifications in the function, structure, and regulation of proteins and protein complexes.

Full text of DOI:10.1021/jacs.8b03572

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Article
Hydrazide Mimics for Protein Lysine Acylation to Assess
Nucleosome Dynamics and Deubiquitinase Action
Shridhar Bhat, Yousang Hwang, Matthew D Gibson, Michael T Morgan, Sean D.
Taverna, Yingming Zhao, Cynthia Wolberger, Michael Guy Poirier, and Philip A Cole
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b03572 • Publication Date (Web): 10 Jul 2018
Downloaded from http://pubs.acs.org on July 10, 2018
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Hydrazide Mimics for Protein Lysine Acylation to Assess
Nucleosome Dynamics and Deubiquitinase Action
†
,¶,#
†,#
‡,◊
§,◊
Shridhar Bhat,
Yousang Hwang, Matthew D. Gibson, Michael T. Morgan, Sean D.┴
ǁ
§ *, ‡
Taverna, Yingming Zhao, Cynthia Wolberger, Michael G. Poirier, and Philip A. Cole*,†,
†
,¶
†Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205, USA.
¶Center for Epigenetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA
‡Department of Physics, Ohio State University, Columbus, Ohio 43210, USA.
§Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21205, USA.
ǁ
Ben May Department for Cancer Research, The University of Chicago, Chicago, IL 60637, USA
┴
Division of Genetics, Brigham and Women’s Hospital; Departments of Medicine and Biological Chemistry and
Molecular Pharmacology, Harvard Medical School, 77 Ave Louis Pasteur, HMS New Research Building, Boston,
Massachusetts 02115, USA.
ABSTRACT: A range of acylꢀlysine (acylꢀLys) modifications on histones and other proteins have been mapped over
the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in
the study of acylꢀLys containing proteins is the challenge of producing them or their mimics in siteꢀspecifically modified
forms. We describe a cysteine alkylationꢀbased method to install hydrazide mimics of acylꢀLys postꢀtranslational
modifications (PTMs) on proteins. We have applied this method to install mimics of acetylꢀLys, 2ꢀhydroxyisobutyrylꢀ
Lys, and ubiquitylꢀLys that could be recognized selectively by relevant acylꢀLys modification antibodies. The acylꢀLys
modified histone H3 proteins were reconstituted into nucleosomes to study nucleosome dynamics and stability as a
function of modification type and site. We also installed a ubiquitylꢀLys mimic in histone H2B and generated a
diubiquitin analog, both of which could be cleaved by deubiquitinating enzymes. Nucleosomes containing the H2B
ubiquitylꢀLys mimic were used to study the SAGA deubiquitinating module’s molecular recognition. These results
suggest that acylꢀLys mimics offer a relatively simple and promising strategy to study the role of acylꢀLys modifications
in
the
function,
structure,
and
regulation
of
proteins
and
protein
complexes.


INTRODUCTION
Lysine sidechain modifications on histones were
siteꢀdirected mutagenesis has been used to investigate
the roles of specific acetylꢀLys modifications by
introducing glutamine as a mimic, but the results can be
difficult to interpret.4
1a
discovered about 50 years ago. Since then, these
reversible covalent marks have been shown to be
important for the regulation of genome processing
including gene expression and DNA repair. The last
decade has seen the discovery of a plethora of novel postꢀ
translational modifications (PTMs) involving the
acylation of the lysine sidechain (acylꢀLys). Thousands
of lysine acetylation and ubiquitylation sites have been
mapped and increasing numbers of novel acylꢀLys
modifications such as 2ꢀhydroxyisobutyrylation (hib)
have been identified (Figure 1A).
functions of the vast majority of these lysine
modifications, however, have remained elusive. To
determine the functions of such acylꢀLys PTMs, it is
important to have proteins containing stoichiometric and
siteꢀspecific modifications or faithful mimics. Standard
Several elegant strategies have been developed for
installing acylꢀLys modifications and their mimics into
1
5
proteins, including total chemical synthesis, chemical
6
7
ligation strategies, nonsense suppression mutagenesis,
2
and cysteine (Cys) modifications8a,9,10 but the technical
complexity, scope, or yields have so far limited their
applications. Total chemical synthesis of proteins
containing ubiquitylation and acetylation has been
3
shown to be effective for small proteins11 but because of
The biological
technical challenges, this method is out of reach for most
biochemistry labs.
Expressed protein ligation and
related semisynthetic methods have been used to
6c,12
13
introduce acetylꢀ
into proteins, but these methods have typically been
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Journal of the American Chemical Society
employed to install modifications at the termini of a Here we investigate a new Cys modification strategy
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protein of interest.
The use of nonsense codon
that can install hydrazide analogs of acylꢀLys mimics.
We show that this relatively simple semisynthetic
method can be used to install small modifications into
proteins such as an acetylꢀLys mimic, as well as larger
modifications such as a ubiquitylꢀLys (Figure 1C). We
examine the recognition of these acylꢀLys mimics using
antibodies and their macromolecular effects regarding
suppression to incorporate acylꢀLys has shown promise,
but has largely been used in prokaryotic expression
systems, the yields can be variable, and enzymatic
deacylation during isolation is a concern. The unique
reactivity of the Cys sulfhydryl group in a protein has
been harnessed before to install good mimics of methylꢀ
7c
15a
10b,15b
8a,9,10
arginine,
Figure 1B) modifications. The key step in the mimic
installation in these methods is either a chemoselective
methylꢀlysine,
and acylꢀlysine
nucleosome
dynamics
and
recognition
by
(
deubiquitinases (DUBs) and describe these studies
below.
8
a,15
9,10
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alkylation of the thiol
or a thiol–ene reaction of the
Cys sideꢀchain, where the latter also includes a variation
of Cys to dehydroalanine conversion followed by thiol–
ene reaction with a modification bearing thiol.1 Thus,
all these mimics contain a sulfur atom instead of the γꢀ
methylene, but that minor difference has not proven
detrimental to the mimicry.

EXPERIMENTAL METHODS
Acid hydrazides. Acetohydrazide was purchased from
0b
19a
SigmaꢀAldrich. 2ꢀHydroxyꢀisobutyric acid hydrazide
19b
and ubiquitin hydrazide have been reported previously
and our method of preparation is delineated in the
Supporting Information.
Ubiquitylation, a PTM that results in an appendage of
.5 kDa, can be regarded as an especially challenging
Expression and purification of histones. Protocols for
bacterial overexpression and purification of H3ꢀK56C,
8
target among acylꢀLys modifications. Cys modification
H3ꢀK122C (these K
H3.1 harboring C96S, G102A, and C110A mutations),
H2BꢀK120C, H2BꢀK116A,K120C (these mutants
→C mutants are derived from human
10,13
16
methods,
disulfide
linkage,
azideꢀalkyne
17
5aꢀc
cycloaddition, total chemical synthesis, and nonsense
suppression mutagenesis followed by traceless ligation
18
originated from truncated, aa4–125, Xenopus laevis
H2Bꢀ1.1) and other core histones are detailed in the
Supporting Information.
have all been shown to have utility in the production of
ubiquitylꢀprotein conjugates. Perhaps because of either
technically challenging chemical manipulations in some
cases or the significant departure from the native
structures in others, however, these strategies have not
yet been widely adopted. As there are tens of thousands
of acylꢀLys sites that have been mapped, simple and
efficient methods are needed to more rapidly interrogate
the impact of these PTMs.
Installation of hydrazide mimics into histones and
ubiquitin. Lyophilized powder of H3ꢀK56C or K122C (10
mg, final concentration: ~1.0 mM) was dissolved in 100
mM HEPES (added from 1 M stock, pH 8.0) containing
50 mM reducing agent, tris(2ꢀcarboxyethyl)phosphine
(TCEP; added from 0.5 M stock, pH 7.0) and 4.5 M
guanidinium hydrochloride. Chloroacetaldehyde (50%
w/v soln.; final concentration: 15 mM) was then added
and stirred vigorously at room temperature for 30–45
min. The reaction mixture was transferred to a 15 mL
centrifugal filterꢀunit (Amicon Ultraꢀ15, 3 kDa cutoff,
MilliporeSigma) and filled to the brim with dH₂ O and
spun at 4,000 rpm until the volume was reduced to
about 3 mL. This operation was repeated twice by filling
the filter unit with acetate buffer (1 M AcOH + 1 M
NaOAc, pH 4.5). At this stage the filter unit was filled
with acetate buffer containing acylꢀhydrazide (20 mM in
the
case
of
acetohydrazide
and
2ꢀ
hydroxyisobutyrohydrazide, and 10 mM for ubiquitin
hydrazide [UbꢀHz]) and the procedure was repeated. The
final volume of about 1 mL was then transferred to a 5
mL tube and supplemented with 15 mM acylꢀhydrazide
(
this is not needed in the case of UbꢀHz, because, it is
retained during Amicon ultrafiltration) and the mixture
was vigorously stirred at room temperature for 14–18 h.
The final step in this threeꢀstep protocol is the reduction
of hydrazone to hydrazide which is achieved by adding
sodium cyanoborohydride (final concentration: 50 mM)
in 4 M urea and stirring vigorously at room temperature
for 2 h. This reaction mixture can be directly injected (if
not ready for HPLC right away, the sample should be rid
of urea by Amicon ultrafiltration) onto a PROTO 300 C18
column on HPLC (4.6×250 mm, 5µ, 300 Å; Higgins
Analytical) and eluted with acetonitrile/H₂ O (gradient
ramp of 25% to 85% acetonitrile over 40 min) containing
0.05% trifluoroacetic acid (TFA). The HPLC fractions
were examined by ESIꢀMS, purified fractions containing
desired protein species were pooled, concentrated and
lyophilized to obtain the desired histoneꢀPTM mimics as
Figure 1. Lysine sidechain modifications. (A) Naturally
occurring acylꢀLys PTMs. (B) Previously reported acetylꢀLys
9a,8a,8b
mimics.
(C) Threeꢀstep protocol presented in this
manuscript to install acylꢀLys mimics into proteins.
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powders and stored at −80 °C. It should be noted that we
to deplete chloroacetaldehyde but the near complete
modification of the cysteine sulfhydryl group suggests
that this side reaction is inconsequential under our
reaction conditions. We performed these reactions on
histones and ubiquitin engineered to have a single Cys at
the Lys modification site of interest. Moving from
peptides to histones and ubiquitin, the use of 4.5 M
guanidine hydrochloride as chaotrope was found to be
beneficial in the alkylation step. However, the chaotrope
impeded the hydrazone formation, and we found acetate
buffer at pH 4.5 to be conducive to this process, and
hence a buffer exchange was incorporated at this step.
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installed ubiquitin hydrazide mimics on H2BꢀK120C,
H2BꢀK116A,K120C, and ubiquitinꢀK48C (makes K48ꢀ
diubiqutin mimic) using the procedure described above.
Starting from 10 mg of histone, yields of pure histone
PTM mimics ranged between 2–6 mg.
Note: Henceforth, we will use shorthand designations
for acylꢀLys mimics. For example, H3ꢀK 56ac will stand
c
for histone H3 bearing acetylꢀLys hydrazide mimic at
position 56, where the mimic descended from Cys.
H3-K 56ac. Average Mass (Mav), calculated: 15,313.8;
c
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measured, ESIꢀMS: 15,315.5 ± 4.6.
H3-K 56hib.
M
av
,
calculated: 15,357.9; measured,
c
MALDIꢀTOF: 15,357.5, ESIꢀMS: 15,359.5 ± 3.0.
H3-K 56ub. Mav, calculated: 23,810.5; measured, ESIꢀ
c
MS: 23,814.5 ± 4.7.
H3-K 122ac. Mav, calculated: 15,313.8; measured, ESIꢀ
c
MS: 15,316.4 ± 4.1.
H3-K 122hib.
av
M , calculated: 15,357.9; measured,
c
MALDIꢀTOF: 15,356.6, ESIꢀMS: 15,357.2 ± 3.8.
H3-K 122ub. Mav, calculated: 23,810.5; measured, ESIꢀ
c
MS: 23,813.5 ± 3.9.
H2B-K 120ub. Mav, calculated: 22,065.5; measured, ESIꢀ
c
MS: 22,067.5 ± 3.1.
H2B-K116A,K 120ub.
M
av
,
calculated:
22,008.4;
c
measured, ESIꢀMS: 22,012.5 ± 4.2
Kc48-diUbiquitin. Mav, calculated: 17,128.6; measured,
MALDIꢀTOF: 17,132.4.
Immunoblotting, nucleosome reconstitutions, and
subsequent assays including deubiquitination. These
experiments were conducted according to known
methods and are described in the Supporting
Information.


RESULTS AND DISCUSSION.
Synthetic approach to acyl-Lys mimic incorporation. The
goal of this work was to generate acylꢀLys mimics in a
chemically simple way that would be compatible with
recapitulating at least some of the properties of acylꢀLys
modifications. In this regard, we set our sights on
thioetherꢀcontaining acylꢀhydrazide mimics of acylꢀLys
(
Figure 1C, 6). Such acylꢀLys mimics substitute a sulfur
atom for the γꢀmethylene in the Lys side chain and
contain an additional nitrogen atom in the connection
between the εꢀamino group and the carbonyl. Prior
8
a,9,10b
studies on Lys thioether derivatives
and on
8b
hydrazide analogs (Figure 1B) indicated that such
functionality could preserve at least some of the
structural and biochemical features of the corresponding
acylꢀLys.
The general synthetic approach toward these acylꢀLys
analogs pursued here involves a threeꢀstep chemical
sequence starting with a Cysꢀcontaining peptide (model
peptide reaction detailed in Supporting Information) or
protein: thiol alkylation with chloroacetaldehyde
followed by reaction with the corresponding acylꢀ
hydrazide and then sodium cyanoborohydride reduction
Figure 2. Representative characterization data for
(
Figure 1C). During optimization of chloroacetaldehyde
hydrazide mimics (data shown for H3ꢀK
of the hydrazide mimics are in Supplementary Figures). (A)
HPLC trace, peak ‘a’ corresponds to H3ꢀK 56hib and after
c
56hib, remainder
alkylation of cysteines on model peptides and proteins,
we observed that the presence of up to 50 mM TCEP is
tolerated and necessary to drive the reaction to
c
ESIꢀMS analysis ‘b’ was attributed to the minor side product
7, shown in Fig. 1C. (B) ESIꢀMS of HPLC fractions
2
0
completion. TCEP can theoretically undergo alkylation
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corresponding to peak ‘a’. (C) Deconvolution of ESIꢀMS
spectra shown in Fig. 2B above. Reported here are the
Average Masses (Mav).
CB: Colloidal Blue visualization of the loading control). (B)
Pan 2ꢀhydroxyisobutyrylꢀLys antibody. (C) Ubiquitin
antibody. H3ꢀunmod refers to the unmodified human H3.1
harboring C96S, G102A, and C110A mutations. Full gels of
loading controls stained with Colloidal Blue corresponding
to Figure 3A–C are shown in Supplementary Figure 10A–C,
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It should be noted that hydrazone formation is likely a
slower and reversible step (requires ~14 hours to reach
near completion).
This would explain that after
respectively.
Panꢀ2ꢀhydroxyisobutyrylꢀLys
antibody
cyanoborohydride reduction besides the desired PTM
mimic we do observe as a minor side product, separable
on HPLC, hydroxyethyl modified proteins presumably
resulting from aldehyde to alcohol reduction (Figure 1C,
recognizes the modification in H3 better at Kc122 than at
Kc56, and that same observation was made in each
replicate.
7
). This approach was used to incorporate acetyl, 2ꢀ
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hydroxyisobutyryl, and ubiquityl mimics into proteins.
In general, the threeꢀstep process proceeds to ~50ꢀ70%
conversion and final modified protein products are
purified by reversedꢀphase chromatography and
structural confirmation performed using mass
spectrometry (Figure 2).
Effects of acyl-Lys on nucleosome dynamics and stability.
Given the frequency and diversity of histone Lys
modifications and their potential importance in
influencing nucleosome dynamics, we decided to
investigate the effects of acylꢀLys mimics in the context
of histones. We incorporated three acylꢀLys mimics for
acetyl (ac), hydroxyisobutyryl (hib), and ubiquityl (ub) at
sites of histone modifications that were identified in
C
1ꢀ3
proteomics studies. The sites studied here in histone
H3 include H3ꢀK56 (ac, hib, and ub) and H3ꢀK122 (ac,
hib, and ub). These sites were chosen in part because
they occur in the globular region of histone H3, where
they are in proximity to the DNA and therefore may be
more likely to affect nucleosome stability or dynamics.
H3ꢀK56 and H3ꢀK122 acetylation have been shown
previously to influence nucleosome biophysical
2
1
properties.
As one test of whether the acylꢀLys mimics
recapitulated properties of native acylꢀLys modifications,
we used Western blotting to test whether antibodies
specific for particular acylꢀLys modifications would
recognize the acylꢀLys mimics. As shown in Figure 3, the
hydrazide mimics installed on histone H3 (H3ꢀK 56ac,
c
hib, and ub) were selectively recognized by commercial
antibodies that correspond to the appropriate acylꢀLys
modification. These results are consistent with the
designed structural similarity between the acylꢀLys
mimics and the genuine modifications.
D
Figure 3. Selective antibody recognition of hydrazide
mimics. (A) Antibody against H3ꢀK56ac (WB: immunoblot;
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Figure 4. Native PAGE of reconstituted nucleosomes
bearing hydrazide mimics (NCP stands for nucleosome core
particle). Two types of nucleosomes we prepared to study
H3ꢀK56 position. Rather, a combination of polarity,
hydrogen bonding, and other atomic features are
apparently important in mediating the effects of H3ꢀK56
modifications on nucleosome DNA unwrapping.
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their dynamics: NCP
dsDNA and standard NCP which does not include LexA
dsDNA. (A) Nucleosomes with H3ꢀK 56 modifications. (B)
Nucleosomes containing H3ꢀK 122 modifications. (C and D)
L
with LexA target sequence in the
c
c
22
Structures of NCPs (PDB ID: 1KX5), depicting the location
of fluorophores and PTM mimics. Both copies of H3 are
rendered in blue. Locations of Cy3 (green), Cy5 (red), and
PTM mimics are in shown as ‘spheres’. (C) Nucleosomes
used in the LexA binding studies show LexA target sequence
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(
(
yellow ‘sticks’), H3ꢀK56 (orange), and H3ꢀK122 (magenta).
D) NCPs used in salt titrations and kinetics show H3ꢀK122
residues in orange. H3ꢀunmod (as described in Figure 3
legend above) was used to assemble both NCP (unmod) and
L
NCP(unmod) in Figures 4A and 4B, respectively. Also, these
NCPs were loaded on every third lane to help reveal the gel
shifts due to PTMs. This observation has been noted in
previous reports (see Fig. 3cꢀd in reference 5d or Fig. 2A in
reference 21c).
One potential consequence of acylꢀLys modifications in
the context of histones is that they may loosen
nucleosome structure. Indeed, prior work on acetylation
of histone H3 on Lys56, a modification seen in DNA
damage in yeast, has shown that this modification can
enhance nucleosome DNA unwrapping in the entry/exit
5d
region of the nucleosome. We therefore proceeded to
compare the effects of the various histone H3
modifications on nucleosome DNA unwrapping. We
assembled nucleosomes containing a LexA protein
binding site in
a Widom 601 DNA nucleosome
positioning sequence (LexA site bp 8ꢀ27) that also
included Cy3 and Cy5 fluorescent tags at DNA bp 1 and
histone H2AꢀK119C, respectively (Figure 4A and 4C).
Cy3 is a FRET donor to Cy5, so when LexA binds to its
canonical site in the nucleosome, it traps the nucleosome
in a partially unwrapped state increasing the Cy3–Cy5
distance and decreasing the FRET signal. The affinity of
LexA for these nucleosomes can therefore be measured
by monitoring the FRET change (Figure 5A).
Figure 5. Effect of H3ꢀK56 acyl modifications on
nucleosome dynamics. (A) Titration of LexA against
unmodified (comprising H3ꢀunmod) and modified
nucleosomes bearing hydrazide mimics on H3ꢀK56. (B)
Tabulation of S½ values (concentration at which half of the
nucleosomes are bound by LexA) and relative DNA
accessibilities of nucleosomes for LexA binding with
It had previously been shown that H3ꢀK56 acetylation
induces a ~3ꢀfold enhanced affinity for LexA in this
assay, correlating with enhanced nucleosome DNA
different H3ꢀK56 acyl mimics. *Prior data on natural acylꢀ
5d
Lys5d; **previous5d S½ was normalized relative to the
unwrapping with this PTM. Here we show that the
acetylꢀLys hydrazide mimic at the H3ꢀK56 position
increases LexA affinity by 2ꢀfold, closely agreeing with
L
current S½ of NCP (unmod).
We also explored the effects of H3ꢀK122 acylꢀLys
mimics on nucleosome stability. For these studies, FRET
of the intact nucleosome was assessed using DNA labeled
with Cy3 at bp 58 and histone H4 tagged at the 21
position (V21C) with Cy5 which can report on the
nucleosome dyad symmetry axis. This dyad region
shows dense histoneꢀDNA interactions and acetylation of
5d
the effect of the natural acetylꢀLys. In contrast to the
effects of H3ꢀK56 acetylꢀLys mimic, H3ꢀK122
modification by the acetylꢀLys mimic had essentially no
influence
on
nucleosome
DNA
unwrapping
(
Supplementary Figure 11). These results are consistent
with the expectation that H3 PTMs distal from the
nucleosome entry/exit sites do not enhance unwrapping
and that the chemical manipulation of H3 per se is not a
source of nonꢀspecific effects. Moreover, the H3ꢀK56hib
and H3ꢀK56ub hydrazide mimics increased LexA binding
by 12ꢀfold and 6ꢀfold, respectively (Figure 5B). These
findings suggest that both the hydroxyisobutyrylation
and ubiquitylation of H3ꢀK56 show a heightened ability
to enhance nucleosome DNA unwrapping, perhaps
because of their larger size as compared to acetylation.
That the significantly bigger ubiquitylation does not
perturb unwrapping more than hydroxyisobutyrylation
indicates that PTM size per se is not the only factor to
influence nucleosome DNA unwrapping at the histone
23
H3ꢀK122 can enhance remodeling by SWI/SNF. Here
we found that incorporation of the acetyl,
hydroxyisobutyryl
modifications each showed similar destabilizing effects of
the nucleosome as function of increasing salt
and
ubiquityl
hydrazideꢀLys
a
concentration (Figure 6A). Corroborating these findings,
the rate of nucleosome denaturation was faster at a fixed
ionic strength (Figure 6B). These results suggest that
acyl modification of the natural Lys side chain at H3ꢀ
K122, regardless of bulk of the substitution, is sufficient
to destabilize nucleosomes in this region (Figure 6C).
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Figure 7. Deubiquitinase reactions of (A) Ubp10, (B)
Ubp8/SAGA DUB module, and (C) hOTUB1 with natural
and hydrazide mimic versions of K48ꢀdiubiquitin.
Figure 6. Effect of H3ꢀK122 modifications on nucleosome
stability. (A) Normalized FRET response due to a titration of
NaCl in presence of nucleosomes with and without acylꢀLys
mimics at H3ꢀK122. (B) Decay of FRET acceptor response
through donor excitation on nucleosomes with and without
acylꢀLys modifications at H3ꢀK122 at a fixed concentration
of salt (1,125 mM). (C) Tabulation of results from salt
titration and kinetics.
Ubp8 functions as part of a DUB module in the yeast
SAGA complex which is
comprising the proteins, Ubp8, Sgf11, Sus1, and Sgf73.
a multiꢀprotein complex
2
6
An Xꢀray crystal structure has revealed the architecture
of the complex formed by the DUB module with a
2
7
ubiquitinated nucleosome. Like many DUBs, Ubp10,
Ubp8, and OTUB1 are Cys hydrolase enzymes in which
the active Cys serves as a nucleophile that attacks the
isopeptide (amide) carbonyl carbon of the ubiquitin
attached to a Lys sidechain in protein substrates.
Subsequent hydrolysis of the CysꢀUb thioester affords
free ubiquitin and regenerates the unmodified DUB
catalyst.
Ubiquityl-Hydrazide Lys mimics as deubiquitinase
substrates. The molecular recognition of ubiquityl
hydrazide analogs was probed with three purified
deubiquitinase (DUB) enzymes: yeast Ubp10, yeast
Ubp8, and human OTUB1. Ubp10 and Ubp8 have been
implicated in the removal of ubiquitin from Lys123 in
For the initial experiments, we prepared the
corresponding diubiquitin substrates linked at the Lys48
position in ubiquitin via the Cys modification strategy
described above. The ability of Ubp10, Ubp8/SAGA DUB
module, and OTUB1 to cleave the diubiquitin hydrazide
analogs was compared with cleavage of K48ꢀlinked
2
4
histone H2B whereas OTUB1 is reported to cleave K48ꢀ
2
5
linked polyubiquitin and stabilize the transcription
factor FOXM1.
diubiquitin containing
experiments, we used fixed reaction times and
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Page 7 of 9
Journal of the American Chemical Society
diubiquitin protein substrate along with various
modified nucleosomes with Ubp8/SAGA DUB module
showed that both substrates were rapidly processed with
approximately equal rates (Figure 8). Overall, these
studies suggest that Lys116 in ubiquitylated H2B does
not make critical contacts for Ubp8/SAGA DUB module
binding and turnover.
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concentrations of DUB. The reaction conditions were
based on the specific reactivity of the particular DUB
with natural K48ꢀlinked diubiquitin. We found that the
diubiquitin hydrazide analog was readily cleaved by each
of the three DUBs analyzed, suggesting that the
diubiquitin hydrazides were recognized in an
enzymatically relevant way by the enzymes.
Interestingly, Ubp10 cleaved the diubiquitin hydrazide
substrate about 10ꢀfold more efficiently than the natural
diubiquitin substrate (Figure 7A) and the Ubp8 SAGA
DUB module processed diubiquitin hydrazide about 3ꢀ
fold more efficiently than natural diubiquitin (Figure

CONCLUSION
Here we have developed a new approach to installing
acylꢀLys mimics at specific sites in proteins by Cys
modification. The chemical steps are relatively simple to
execute and can readily be performed on histones and
ubiquitin. Like other Cys alkylation approaches, it is
necessary to replace natural Cys in a protein of interest to
avoid nonꢀtargeted mutations. Although we have thus
far applied this method to relatively small proteins that
can be denatured and refolded, the lack of a requirement
for organic solvent suggests that it should be possible to
adapt this method to a wide range of larger proteins of
interest without the need for denaturation.
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B). By contrast, OTUB1 cleaved diubiquitin hydrazide
about 3ꢀfold less efficiently than natural diubiquitin
substrate (Figure 7C).
The sulfur for methylene substitution and the extra
nitrogen in the acylꢀLys mimics used here seem to be
tolerated in antibody recognition and in the case of
ubiquitin, DUB processing. The latter feature might
make them particularly useful mechanistic tools for the
study of DUB substrate selectivity and structural
interactions. As with all mimics, there will undoubtedly
be cases where the differences of these mimics from the
natural functionalities will alter biochemical behaviors.
Nevertheless, we expect that the chemical ease for
generating these mimics will allow them to be
investigated in a range of settings to explore the structure
and function of acylꢀLys protein modifications.
Figure 8. Deubiquitination of nucleosomes bearing H2Bꢀ
K
c
120ub and H2BꢀK116AꢀK
c
120ub by Ubp8/SAGA DUB
module.
These results indicate that, for these three DUBs, the
hydrazide mimic is wellꢀrecognized by the enzymes in
terms of binding and catalysis. However, there appear to
be unique features for each of the enzymes that influence
the precise rate of processing. Since cleavage rates on
the hydrazide mimic were higher for the two USP class
DUBs, Ubp8 and Ubp10, and lower for OTUB1, a
member of the OTU class of DUBs, it is possible that
unique features near the active site influence reactivity of
the hydrazide mimic. We speculate that the increased
cleavage observed with Ubp10 and Ubp8 on the
diubiquitin hydrazide substrate relative to the natural
substrate may be because of the lower pKa of the
hydrazine leaving group relative to an amine leaving
group, although further mechanistic studies will be
needed to evaluate this.


ASSOCIATED CONTENT
SUPPORTING INFORMATION.
Preparation of 2ꢀhydroxyisobutyric hydrazide and its
installation as a 2ꢀhydroxyisobutyrlꢀLys mimic onto a model
peptide.
Preparation of ubiquitin hydrazide, K
→C mutant H3 and
H2B histones, core histones, other proteins used in the
assays, and DNA constructs.
Details of immunoblotting experiments.
Histone octamer refolding and nucleosome reconstitutions.
Site accessibility and stability assays with nucleosomes.
Production and purification of Ubp10, hOTUB1,
Ubp8/SAGA DUB module and deubiquitination assays.
Selected HPLC traces, MALDIꢀTOF, ESIꢀMS, and the
corresponding deconvolution spectra of hydrazide mimics of
model peptide, H3, H2B, and diubiquitin.
We next explored the wellꢀestablished Ubp8/SAGA
DUB module's enzymatic function by analyzing the H2Bꢀ
K120ꢀubiquitylated (H2BꢀmonoUb at K123 in yeast is
equivalent to K120 in Xenopus laevis or human)
hydrazide nucleosome substrate. Prior elucidation of the
Ubp8/SAGA DUB module's structural interactions with
an H2B K120ꢀubiquitylated nucleosome used a histone
Full gels of the loading controls corresponding to Figures
3
A–C.
LexA—nucleosome binding isotherms comprising a negative
control (NCP with H3ꢀK 122ac).
c
This material is available free of charge via the Internet at
http://pubs.acs.org.
H2B that contained
a K116A mutation that was
◾◾ AUTHOR INFORMATION
2
7
introduced to simplify H2BꢀK120Ub synthesis. It was
formally possible that Lys116 might influence
Ubp8/SAGA DUB module recognition of the K120ꢀ
ubiquitylated nucleosome substrate, which was not
investigated previously. We therefore prepared two
forms of Lys120ꢀubiquitin hydrazide histone, H2B
containing either Lys116 or Ala116 and then incorporated
these modified H2Bs into nucleosomes. Reaction of these
Corresponding Authors
*
poirier.18@osu.edu, pacole@bwh.harvard.edu
Author Contributions
#S. Bhat and Y. Hwang contributed equally to this work. ◊M.
Gibson and M. Morgan contributed equally to this work.
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Page 8 of 9
G.; Bao, X.; Ishibashi, T.; Li, X. D. Cell Chem. Biol. 2018, 25(2),
166ꢀ174.
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Notes
(
10) (a) Valkevich, E. M.; Guenette, R. G.; Sanchez, N. A.;
The authors declare no competing financial interest.
Chen, YꢀC.; Ge, Y.; Strieter, E. R. J. Am. Chem. Soc. 2012,
1
34(16), 6916–919. (b) Chalker, J. M.; Lercher, L.; Rose, N. R.;


ACKNOWLEDGMENTS
Schofield, C. J.; Davis, B. G. Angew. Chem. Int. Ed. 2012,
51(8), 1835–839.
We thank the NIGMS GM62437 (P.A.C.), GM095822 (C.W.)
and GM121966 (M.G.P.) and FAMRI Foundation (P.A.C.)
for financial support.
(
(
11) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338–351.
12) (a) Chiang, K. P.; Jensen, M. S.; McGinty, R. K.; Muir, T.
W. ChemBioChem 2009, 10(13), 2182–187. (b) Karukurichi, K.
R.; Wang, L.; Uzasci, L.; Manlandro, C. M.; Wang, Q.; Cole, P.
A. J. Am. Chem. Soc. 2010, 132(14), 1222–223. (c) Nguyen, U.
T. T.; Bittova, L.; Muller, M. M.; Fierz, B.; David, Y.; Houckꢀ
Loomis, B.; Feng, V.; Dann, G. P.; Muir, T. W. Nat. Methods

REFERENCES
(
1) (a) Allfrey, V. G.; Mirsky, A. E. Science 1964, 144(3618),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
559. (b) Li, B.; Carey, M.; Workman, J. L. Cell 2007, 128(4),
7
5
07–719. (c) Cole, P.A. Nature Chem. Biol. 2008, 4(10), 590ꢀ
97. (d) Bannister, A. J.; Kouzarides, T. Cell Res. 2011, 21, 381–
2
014, 11(8), 834–840. (d) Miller, T. C. R.; Simon, B.; Rybin, V.;
Grotsch, H.; Curtet, S.; Khochbin, S.; Carlomagno, T.; Muller, C.
3
95.
2) (a) Lin, H.; Su, X.; He, B. ACS Chem. Biol. 2012, 7(6),
947ꢀ960. (b) Olsen, C. A. ChemMedChem 2014, 9(3), 434–437.
c) Rousseaux, S.; Khochbin, S. Cell J. 2015, 17(1), 1–6. (d)
Huang, H.; Lin, S.; Garcia, B. A.; Zhao, Y. Chem. Rev. 2015,
15(6), 2376–2418. (e) Hirschey, M. D.; Zhao, Y. Mol. Cell.
Proteomics 2015, 14(9), 2308–315. (f) PhosphoSitePlus®
Online Systems Biology Resource.
https://www.phosphosite.org/
3) (a) Dai, L.; Peng, C.; Montellier, E.; Lu, Z.; Chen, Y.; Ishii,
W. Nat. Commun. 2016, 7, 13855.
(
(
13) (a) McGinty, R. K.; Kim, J.; Chatterjee, C.; Roeder, R. G.;
Muir, T. W. Nature 2008, 453(7196), 812–816. (b) Fierz, B.;
Kilic, S.; Hieb, A. R.; Luger, K.; Muir, T. W. J. Am. Chem. Soc.
(
2
012, 134(48), 19548–551.
(14) Schwarzer, D.; Cole, P. A. Curr. Opin. Chem. Biol. 2005,
1
9
(6), 561–569.
15) (a) Le, D. D.; Cortesi, A. T.; Myers, S. A.; Burlingame, A.
L.; Fujimori, D. G. J. Am. Chem. Soc. 2013, 135(8), 2879–82.
b) Simon, M. D.; Chu, F.; Racki, L. R.; de la Cruz, C. C.;
(
(
(
H.; Debernardi, A.; Buchou, T.; Rousseaux, S.; Jin, F.; Sabari, B.
R.; Deng, Z.; Allis, C. D.; Ren, B.; Khochbin, S.; Zhao, Y. Nat.
Chem. Biol. 2014, 10(5), 365–370. (b) Huang, H.; Luo, Z.; Qi,
S.; Huang, J.; Xu, P.; Wang, X.; Gao, L.; Li, F.; Wang, J.; Zhao,
W.; Gu, W.; Chen, Z.; Dai, L.; Dai, J.; Zhao, Y. Cell Res. 2018,
Burlingame, A. L.; Panning, B.; Narliker, G. T.; Shokat, K. M.
Cell 2007, 128(5), 1003ꢀ12.
(
16) (a) Chatterjee, C.; McGinty, R. K.; Fierz, B.; Muir, T. W.
Nat. Chem. Biol. 2010, 6(4), 267–69.
17) (a) Rösner, D.; Schneider, T.; Schneider, D.; Scheffner,
(
2
8(1), 111–125.
M.; Marx, A. Nat. Protoc. 2015, 10(10), 1594–611. (b) Zhao, X.;
Lutz, J.; Hollmuller, E.; Scheffner, M.; Marx, A.; Stengel, F.
Angew. Chem. Int. Ed. 2017, 56(49), 15764–768. (c) Zhang, F.;
Smits, A. H.; van Tilburg, G. B. A.; Jansen, P. W. T. C.;
Makowski, M. M.; Ovaa, H.; Vermeulen, M. Mol. Cell 2017,
65(5), 941–55.
(
4) (a) Wang, YꢀH.; Tsay, YꢀG.; Tan, B. CꢀM.; Lo, WꢀY.; Lee,
SꢀC. J. Biol. Chem. 2003, 278(28), 25568–576. (b) Manohar,
M.; Mooney, A. M.; North, J. A.; Nakkula, R. J.; Picking, J. W.;
Edon, A.; Fishel, R.; Poirier, M. G.; Ottesen, J. J. J. Biol Chem.
2009, 284(35), 23312–321. (c) Wollebo, H. S.; Bellizzi, A.;
Cossari, D. H.; Salkind, J.; Safak, M.; White, M. K.
Neurovirol. 2016, 22(5), 615–625.
J
(
18) (a) Yang, R.; Pasunooti, K. K.; Li, F.; Liu, XꢀW.; Liu, CꢀF.
J. Am. Chem. Soc. 2009, 131(38), 13592–593. (b) Virdee, S.;
Kapadnis, P. B.; Elliott, T.; Lang, K.; Madrzak, J.; Nguyen, D.
P.; Riechmann, L.; Chin, J. W. J. Am. Chem. Soc. 2011,
133(28), 10708–711. (c) Virdee, S.; Ye, Y.; Nguyen, D. P.;
Komander, D.; Chin, J. W. Nat. Chem. Biol. 2010, 6(10), 750–
(5) (a) Kumar, K. S. A.; Spasser, L.; Erlich, L. A.; Bavikar, S.
N.; Brik, A. Angew. Chem. Int. Ed. 2010, 49(48), 9126–131. (b)
El Oualid, F.; Merkx, R.; Ekkebus, R.; Hameed, D. S.; Smit, J.
J.; de Jong, A.; Hilkmann, H.; Sixma, T. K.; Ovaa, H. Angew.
Chem. Int. Ed. 2010, 49(52), 10149–153. (c) Kumar, K. S. A.;
Bavikar, S. N.; Spasser, L.; Moyal, T.; Ohayon, S.; Brik, A.
Angew. Chem. Int. Ed. 2011, 50(27), 6137–141. (d) Shimko, J.
C.; North, J. A.; Bruns, A. N.; Poirier, M. G.; Ottesen, J. J. J.
Mol. Biol. 2011, 408(2), 187–204.
5
7.
(
19) (a) Galons, H.; Cave, C.; Miocque, M.; Rinjard, P.; Tran,
G.; Binet, P. Eur. J. Med. Chem. 1990, 25(9), 785–788. (b) Li,
YꢀT.; Liang, J.; Li, JꢀB.; Fang, GꢀM.; Huang, Y.; Liu, L. J. Pept.
Sci. 2014, 20(2), 102–107.
(
6) (a) Dawson, P.; Muir, T.; ClarkꢀLewis, I.; Kent, S. Science
(
20) Bos, J.; Muir, T. W. J. Am. Chem. Soc. 2018, 140(14),
757–4760.
21) (a) Neumann, H.; Hancock, S. M.; Buning, R.; Routh, A.;
1994, 266(5186), 776–779. (b) Muir, T. W.; Sondhi, D.; Cole, P.
A. Proc. Natl. Acad. Sci. USA 1998, 95(12), 6705–6710. (c)
ShogrenꢀKnaak, M.; Ishii, H.; Sun, JꢀM.; Pazin, M. J.; Davie, J.
R.; Peterson, C. L. Science 2006, 311(5762), 844–847. (d)
McGinty, R. K.; Chatterjee, C.; Muir, T. W. Methods Enzymol.
2009, 462, 225–243.
4
(
Chapman, L.; Somers, J.; OwenꢀHughes, T.; van Noort, J.;
Rhodes, D.; Chin, J. W. Mol. Cell 2009, 36(1), 153–63. (b)
Simon, M.; North, J. A.; Shimko, J. C.; Forties, R. A.;
Ferdinand, M. B.; Manohar, M.; Zhang, M.; Fishel, R.; Ottesen,
J. J.; Poirier, M. G. Proc. Natl. Acad. Sci. USA 2011, 108(31),
(
7) (a) Neumann, H.; PeakꢀChew, S. Y.; Chin, J. W. Nat.
Chem. Biol. 2008, 4(4), 232–234. (b) Wan, W.; Huang, Y.;
Wang, Z.; Russell, W. K.; Pai, PꢀJ.; Russell, D. H.; Liu, W. R.
Angew. Chem. Int. Ed. 2010, 49(18), 3211–214. (c) Gattner, M.
J.; Vrabel, M.; Carell, T. Chem. Commun. 2013, 49, 379–381.
1
2711–716. (c) North, J. A.; Shimko, J. C.; Javaid, S.; Mooney,
A. M.; Shoffner, M. A.; Rose, S. D.; Bundschuh, R.; Fishel, R.;
Ottesen, J. J.; Poirier, M. G. Nucleic Acids Res. 2012, 40(20),
1
0215–227.
22) Davey, C. A.; Sargent, D. F.; Luger, K.; Maeder, A. W.;
(
d) Wang, W. W.; Zeng, Y.; Wu, B.; Deiters, A.; Liu, W. R. ACS
(
Chem. Biol. 2016, 11(7), 1973–981. (e) Wang, Z. A.; Kurra, Y.;
Wang, X.; Zeng, Y.; Lee, YꢀJ.; Sharma, V.; Lin, H.; Dai, S. Y.;
Liu, W. R. Angew. Chem. Int. Ed. 2017, 129(6), 1665–669.
(8) (a) Huang, R.; Holbert, M. A.; Tarrant, M. K. Curtet, S.;
Colquhoun, D. R.; Dancy, B. M.; Dancy, B. C.; Hwang, Y.; Tang,
Y.; Meeth, K.; Marmorstein, R.; Cole, R. N.; Khochbin, S.; Cole,
P. A. J. Am. Chem. Soc. 2010, 132(29), 9986–987. (b) Dancy,
B. C. R.; Ming, S. A.; Papazyan, R.; Jelinek, C. A.; Majumdar, A.;
Sun, Y.; Dancy, B. M.; Drury, W. J. III, Cotter, R. J.; Taverna, S.
D.; Cole, P. A. J. Am. Chem. Soc. 2012, 134(11), 5138–148.
Richmond, T. J. J. Mol. Biol. 2002, 319(5), 1097–1113.
(23) Chatterjee, N.; North, J. A.; Dechassa, M. L.; Manohar,
M.; Prasad, R.; Luger, K.; Ottesen, J. J.; Poirier, M. G.;
Bartholomew, B. Mol. Cell. Biol. 2015, 35(23), 4083ꢀ4092.
(
24) Schulze, J. M.; Hentrich, T.; Nakanishi, S.; Gupta, A.;
Emberly, E.; Shilatifard, A.; Kobor, M. S. Genes Dev., 2011,
25(21), 2242ꢀ2247.
(
25) Wang, T.; Yin, L.; Cooper, E. M.; Lai, M. Y.; Dickey, S.;
Pickart, C. M.; Fushman, D.; Wilkinson, K. D.; Cohen, R. E.;
Wolberger, C. J. Mol. Biol. 2009, 386(4), 1011ꢀ1023.
(
9) (a) Li, F.; Allahverdi, A.; Yang, R.; Lua, G. B. J.; Zhang,
(
26) (a) Henry, K. W.; Wyce, A.; Lo, W. S.; Duggan, L. J.;
X.; Cao, Y.; Korolev, N.; Nordenskiöld, L.; Liu, CꢀF. Angew.
Chem. Int. Ed. 2011, 50(41), 9611–14. (b) Jing, Y.; Liu, Z.; Tian,
Emre, N. C.; Kao, C. F.; Pillus, L.; Shilatifard, A.; Osley, M. A.;
Berger, S. L. Genes Dev. 2003, 17(21), 2648–2663. (b) Kohler,
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
A.; Zimmerman, E.; Schneider, M.; Hurt, E.; Zheng, N. Cell
1
2
3
4
5
6
7
8
9
2010, 141(4), 606–17. (c) Samara, N. L.; Datta, A. B.; Berndsen,
C. E.; Zhang, X.; Yao, T.; Cohen, R. E.; Wolberger, C. Science
2
010, 328(5981), 1025–29.
27) Morgan, M. T.; HajꢀYahya, M.; Ringel, A. E.; Bandi, P.;
Brik, A.; Wolberger, C. Science 2016, 351(6274), 725ꢀ728.
(
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