A Versatile Approach for Site-Specific Lysine Acylation in Proteins

Article abstract of DOI:10.1002/anie.201611415

Using amber suppression in coordination with a mutant pyrrolysyl-tRNA synthetase-tRNA^Pylpair, azidonorleucine is genetically encoded in E. coli. Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site-specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su-H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post-translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.

Full text of DOI:10.1002/anie.201611415

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201611415
German Edition: DOI: 10.1002/ange.201611415
Protein Modifications
Hot Paper
AVersatile Approach for Site-Specific Lysine Acylation in Proteins
Zhipeng A. Wang+, Yadagiri Kurra+, Xin Wang+, Yu Zeng, Yan-Jiun Lee, Vangmayee Sharma,
Hening Lin, Susie Y. Dai,* and Wenshe R. Liu*
Abstract: Using amber suppression in coordination with
a mutant pyrrolysyl-tRNA synthetase-tRNAPyl pair, azidonor-
leucine is genetically encoded in E. coli. Its genetic incorpo-
ration followed by traceless Staudinger ligation with a phos-
phinothioester allows the convenient synthesis of a protein with
a site-specifically installed lysine acylation. By simply changing
the phosphinothioester identity, any lysine acylation type could
be introduced. Using this approach, we demonstrated that both
lysine acetylation and lysine succinylation can be installed
selectively in ubiquitin and synthesized histone H3 with
succinylation at its K4 position (H3K4su). Using an H3K4su-
H4 tetramer as a substrate, we further confirmed that Sirt5 is an
Figure 1. A proposed versatile approach to access proteins with lysine
acylations. A) Post-translationally acylated lysines and their abbrevia-
tions. Acylations with small proteins such as ubiquitin and ubiquitin-
like proteins are not shown. B) Genetic incorporation of AznL followed
active histone desuccinylase. Lysine succinylation is a recently
identified post-translational modification. The reported tech-
nique makes it possible to explicate regulatory functions of this
modification in proteins.
by traceless Staudinger ligation with a phosphinothioester to install
a site-specific lysine acylation in a protein. The reducing nature of the
A
s the only amino acid with a side chain amine, lysine
phosphinothioester leads to the partial conversion of AznL to lysine as
a side product. Shown in the inset are two initially designed phosphi-
nothioesters.
undergoes a myriad of post-translational acylations (Fig-
ure 1A).[1,2] The most studied lysine acylation is acetylation. It
was initially discovered in histones and is widespread in
transcription factors and cytosolic proteins.[3] Two other well-
known lysine acylations are biotinylation and lipoylation.
Both modifications anchor a catalytic cofactor to enzymes.[4]
With the advent of sophisticated proteomic techniques, there
has been an increased diversity of lysine acylations revealed in
histones and non-histone proteins. Three of these novel
acylations, malonylation, succinylation, and glutarylation,
reverse the charge state of the lysine side chain, which
potentiates a unique way of controlling protein functions.[2] So
far, more than ten lysine acylation types have been discov-
ered. However, the functions of novel acylations are largely
unexplored. One impediment is the difficulty of obtaining
proteins with site-specific lysine acylations. The enzymatic
synthesis of proteins with novel lysine acylations is not
applicable since the enzymes responsible for these modifica-
tions are elusive.[5] Alternatively, native chemical ligation/
expressed protein ligation (NCL/EPL), a cysteine-based
alkylation approach, and a recently developed dehydroala-
nine-based coupling approach may be applied for the syn-
thesis of proteins with lysine acylations.[6] However, ECL/
EPL requires a cysteine for ligation and the structures
generated by the latter two methods are slightly different
from the natural modifications. Herein, we report the syn-
thesis of proteins with genuine lysine acylations by coupling
the amber-suppression-based mutagenesis approach[7] with
traceless Staudinger ligation.
[*] Z. A. Wang,[+] Dr. Y. Kurra,[+] Dr. Y. Zeng, Y.-J. Lee, V. Sharma, H. Lin,
Prof. Dr. W. R. Liu
Following the pioneer work by Chin et al., several groups
have successfully engineered the native pyrrolysine incorpo-
ration system for the genetic incorporation of acyl-lysines into
proteins, including Kac, Kpr, Kbu, Kcr, and Khib.[8] Although
convenient, the genetic acyl-lysine incorporation approach
requires that a pyrrolysyl-tRNA synthetase (PylRS) mutant
that recognizes a particular acyl-lysine, but not any native
amino acid, must be identified. The process of identifying
a mutant PylRS is tedious and doesnꢀt always work. So far,
our efforts to find PylRS mutants for Kma, Ksu, and Kgl have
ended with no success. Given the large sizes of Kon, Kbi, Klip,
and Kmy, their direct incorporation at the amber codon is
undoubtedly difficult if not impossible. Inspired by the
traceless Staudinger ligation reaction developed by Bertozzi,
Raines, and others for the conversion of an azide to an acyl
Department of Chemistry, Texas A&M University
College Station, TX 77843 (USA)
E-mail: wliu@chem.tam.edu
Dr. X. Wang,[+] Prof. Dr. S. Y. Dai
Department of Plant Pathology and Microbiology
Institute for Plant Genomics, Office of the Texas State Chemist
Department of Veterinary Pathobiology
College Station, TX 77843 (USA)
E-mail: Susie@ostc.tamu.edu
Prof. Dr. W. R. Liu
Department of Chemistry and Chemical Biology
Cornell University, Ithaca, NY 14853 (USA)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
http://dx.doi.org/10.1002/anie.201611415.
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amide,[9,10] we anticipated that the genetic incorporation of
azidonorleucine (AznL, Figure 1B) followed by a reaction
with a phosphinothioester would effectively combine to
produce a one-size-fits-all approach for the installation of
a large variety of lysine acylations in proteins by simply
changing the acyl group in the phosphinothioester. For
example, using two phosphinothioesters, dPPMT-Ac and
dPPMT-Su, will in theory allow the synthesis of proteins
with site-specific acetylation and succinylation. Although the
intrinsic reductive nature of the phosphinothioester[10,11] will
inevitably convert part of AznL to lysine, this side reaction
leads to the formation of unacylated proteins that could be
resolved from acylated ones using lysine acylation-specific
antibodies for immunoaffinity separation. AznL was previ-
ously installed at the methionine positions of proteins using
the residue-replacement method.[12] Although advantageous
in analyzing nascent proteomes in cells, its global substitution
of methionines makes the approach undesirable for the
purpose of synthesizing proteins with selective lysine acyla-
tions. For this reason, we chose the amber-suppression-based
mutagenesis approach for coding AznL. The synthesis of
AznL followed procedures described in the Supporting
Information, Schemes S1 and S2. A bulk amount around 5 g
was easily made. To identify AznL-specific PylRS mutants,
a PylRS gene library with randomization at five active site
residues, Y306, L309, C348, Y384, and W411, was con-
structed. This library was subjected to a widely adopted
double-sieve selection,[13] yielding a highly efficient AznL-
specific mutant Y306L/C348I/Y384F that is referred to as
AznLRS. For optimal expression, the AznLRS gene was
which agree well with theoretical molecular weights of full-
length sfGFP with AnzL installed at its 134 position
(27866 Da) and its N-terminal methionine cleavage product
(27835 Da) (Figure 2B). The presence of the azide function-
ality in sfGFP-D134AznL was further confirmed by its
selective labeling with an alkyne-fluorescein dye (Supporting
Information, Figure S1). These combined results validated
the specificity of AznLRS toward AznL and the selective
incorporation of AznL at the amber codon.
After demonstrating the selective incorporation of AznL,
we demonstrated its application in the synthesis of proteins
with site-specific acylations. Owing to our ready access to
a MALDI-TOF-MS instrument that is not optimal for
analyzing large proteins such as sfGFP, we switched to work
with ubiquitin. A ubiquitin variant Ub-K48AznL with a C-
terminal 6 ꢁ His tag and AznL incorporated at the original
K48 position was produced similarly as sfGFP-D134AznL.
Expression levels around 10 mgLÀ1 were routinely obtained
(Supporting Information, Figure S2). Ub-K48AznL was then
reacted with excess dMMPT-Ac. DMMPT-Ac and dMMPT-
Su, to be used later, were synthesized according to procedures
described in the Supporting Information, Schemes S3 and S4.
Owing to the slow kinetics of the traceless Staudinger ligation
(a second-order rate constant around 0.001mÀ1 sÀ1),[14] 5 mm
dMMPT-Ac and 378C at pH 6.0 were chosen as the reaction
conditions. Products from different reaction times were
analyzed by SDS-PAGE and then probed by a pan anti-Kac
antibody in the western blot analysis. Ub-K48ac that was
produced using a previously identified AcKRS-tRNAPyl pair
was used as a positive control. The western blot analysis
clearly showed improved acetylation when the incubation
time increased (Figure 3A). The reaction is deemed close to
completion at 48 h since the acetylation level at this reaction
time was not significantly higher than at 36 h. In contrast to
the intense acetylation detected in the reaction products of
Ub-K48AznL, a similar reaction with wild type Ub did not
yield a detectable acetylation level. This result indicates
a much slower intermolecular S-to-N acyl transfer between
dMMPT-Ac and Ub than the traceless Staudinger ligation,
assuring the selectivity of using traceless Staudinger ligation
to convert AznL in a protein specifically to Kac. The
conversion of Ub-K48AznL to its corresponding acetylation
product Ub-K48ac was further confirmed with the MALDI-
TOF-MS analysis of the 48-h reaction product of Ub-
K48AznL. The spectrum displayed two major peaks at 9386
and 9428 Da, representing the corresponding Staudinger
reduction product (a wild type 6 ꢁ His-tagged Ub) and the
traceless Staudinger ligation product (Ub-K48ac), whose
theoretical molecular weights are 9387 and 9429 Da, respec-
tively (Figure 3B). In the final products, Ub-K48ac is slightly
more abundant than wild type Ub. To confirm that the
acetylation is at K48, the 48-h reaction product was trypsi-
nized and analyzed by the tandem mass spectrometry (MS/
MS) analysis. The K48ac-containing fragment was clearly
observed. Its further fragmentation clearly indicated the
presence of acetylation at K48 (Figure 3C).
codon-optimized and then, in coordination with tRNAPyl
,
used in the E. coli BL21 cells to drive the expression of full-
length superfolder green fluorescent protein (sfGFP) in which
an amber codon was introduced at the D134 coding position
of its gene. When cells were grown in the LB medium
supplemented with 5 mm AznL, full-length sfGFP with an
expression level of 20 mgLÀ1 was achieved, which was in
marked contrast with the non-detectable full-length sfGFP
expression observed in the absence of AznL (Figure 2A). The
electrospray ionization mass spectrometry (ESI-MS) analysis
of the expressed protein sfGFP-D134AznL displayed one
major peak at 27866 Da and one minor peak at 27735 Da,
Figure 2. Genetic incorporation of AznL into a model protein sfGFP.
A) Site-specific incorporation of AznL into sfGFP at its D134 position
to produce sfGFP-D134AznL. Cells were transformed with two plas-
mids coding genes for AznLRS, tRNAPyl, and sfGFP with an amber
mutation at its D134 position and grown in the LB medium with or
without 5 mm AnzL. B) Deconvoluted ESI-MS spectrum of purified
sfGFP-D134AznL.
Encouraged by the acetylation results, we applied
dPPMT-Su to the conversion of Ub-K48AznL into Ub-
K48su that is succinylated at the K48 position. The reactions
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dMMPT-NB-Su was synthesized according to procedures
in the Supporting Information, Scheme S5. The use of
dMMPT-NB-Su to trigger the conversion of Ub-K48AznL
to Ub-K48su followed a protocol similar to the synthesis of
Ub-K48ac except that there was an additional step of 365 nm
UV treatment for 30 min before the reaction samples were
analyzed by SDS-PAGE and probed by the pan anti-Ksu
antibody. As shown in Figure 5A, the anti-Ksu-probed
Figure 3. Synthesis of Ub-K48ac. A) Using dPPMT-Ac to convert Ub-
K48AznL to Ub-K48ac. Reaction conditions: 20 mm Ub-K48AznL sam-
ples were incubated with 5 mm dPPMT-Ac in a PBS-buffered H2O/
DMSO (1:1) solution (pH 6.0) at 378C for several hours. After
reactions, the samples were analyzed by SDS-PAGE and probed by
a pan-anti-Kac antibody by western blot. A control reaction with wild
type (wt) Ub was carried out and analyzed similarly. Ub-K48ac was
provided as a positive control. B) MALDI-TOF-MS spectrum of the
reaction product of Ub-K48AznL after its 48-h incubation with dMMPT-
Ac. C) Tandem MS analysis of the trypsinized Kac-containing fragment
of the 48-h reaction product of Ub-K48AznL.
Figure 5. Synthesis of Ub-K48su. A) Using dPPMT-NB-Su to convert
Ub-K48AznL to Ub-K48su. Reaction conditions were similar to the
synthesis of Ub-K48ac except for an additional 365 nm UV treatment
step. B) MALDI-TOF-MS of the 48 h reaction and UV-treated product
of Ub-K48AznL. C) Tandem MS spectrum of the trypsinized Ksu-
containing fragment of the 48 h reaction and UV-treated product of
Ub-K48AznL.
were set up almost identically as for the synthesis of Ub-
K48ac. Products at different reaction times were analyzed by
SDS-PAGE and probed by a pan anti-Ksu antibody. Although
the succinylation on Ub-K48AznL was clearly detected, the
control reaction with wild type Ub also exhibited a high level
of lysine succinylation (Figure 4A), indicating non-selective
succinylation of seven Ub lysines by dPPMT-Su. Since
dPPMT-Su has a carboxylate that potentially triggers an
intramolecular S-to-O acyl transfer reaction with the thioester
to form more reactive succinic anhydride for succinylating Ub
lysines, this result was not a total surprise (Figure 4B).
Indeed, we observed that dPPMT-Su in an oil form was
completely eliminated to succinic anhydride after storage at
À208C for several weeks. To avoid the formation of succinic
anhydride, we conceived dPPMT-NB-Su (Figure 4C). In
dPPMT-NB-Su, a photocleavable nitrobenzyl group[15] shields
the carboxylate from the intramolecular S-to-O acyl transfer
reaction and also allows its easy recovery by UV photolysis.
succinylation on Ub-K48AznL was time dependent and
appeared to reach completion at 48 h. Although the control
reaction with wild type Ub did show detectable lysine
succinylation, its level is minimal. Since we did not observe
a similar side reaction with dMMPT-Ac, it is possible that the
electron-withdrawing inductive effect of the ester in dMMPT-
NB-Su activates its thioester for more favorable S-to-N acyl
transfer with Ub lysines than dMMPT-Ac. To alternatively
confirm the formation of Ub-K48su, the 48-h incubation and
UV treated product of Ub-K48AznL was analyzed by
MALDI-TOF-MS. The spectrum exhibited two major
peaks, one at 9388 Da and the other at 9488 Da, representing
the corresponding wild type Ub and Ub-K48su, whose
theoretic molecular weights are 9387 and 9487 Da, respec-
tively (Figure 5B). Wild type Ub is a little more abundant
than Ub-K48su. To confirm the succinylation at the K48
position, the final product was further trypsinized and
analyzed by tandem MS analysis. The K48su-containing
fragment was detected. Its fragmentation clearly showed
succinylation at K48 (Figure 5C).
Similarly as lysine acetylation, lysine succinylation has
been discovered in histones.[16] Since lysine succinylation
reverses the charge state of lysine, its occurrence in histones is
expected to substantially impact interactions between the
histone octamer and its associated DNA. Sirt5 is a NAD-
dependent protein deacylase that has been suspected to
Figure 4. Nonselective succinylation by dPPMT-Su. A) Ub-K48AznL
reactions with dPPMT-Su. Reactions conditions were similar to the
synthesis of Ub-K48ac. B) An intramolecular S-to-O acyl tranfer reac-
tion of dMMPT-Su. C) Structure of dPPMT-NB-Su.
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remove succinylation from histones and regulate chromatin
functions.[17] To demonstrate Sirt5 is an active histone
desuccinylase, we decided to use the above-demonstrated
approach to synthesize histone H3 with succinylation at K4
(H3K4su) and its tetramer with H4 as a substrate of Sirt5 for
its activity test. H3 with AznL incorporated at K4
(H3K4AznL) was synthesized similarly as Ub-K48AznL. Its
reaction with dMMPT-NB-Su was carried out similarly as for
the synthesis of Ub-K48su. As shown in Figure 6A,
ered novel lysine acylation. Its proteomes have been profiled
in E. coli, Saccharomyces cerevisiae, Toxoplasma gondii,
Vibrio parahemolyticus, Mycobacterium tuberculosis, human
cells, and mouse liver tissues.[18] Many proteins in these
organisms that undergo lysine succinylation are metabolic
enzymes. Their catalytic alteration by lysine succinylation is
anticipated. Xie et al. recently reported lysine succinylation
on histone proteins.[16] One key feature of lysine succinylation
of histones is that it mainly takes place on lysines in the
nucleosome core region. The majority of these lysines involve
direct charge–charge interactions with DNA. Their succiny-
lation not only removes these charge–charge interactions but
also creates repulsion interactions with DNA that loosen the
nucleosome structure. Whether or not cells use this strategy to
unwrap DNA and Sirt5 for its reversal is an interesting aspect
to explore. With our current method, many questions related
to lysine succinylation can be addressed. Since the method
potentiates the synthesis of proteins with any lysine acylation
type, it provides a myriad of opportunities to understand the
functional roles of other novel posttranslational lysine
acylations such as malonylation, glutarylation, and myristoy-
lation. Although not explored in the current study, the
method reported here could be further improved by using
water-soluble phosphinothioesters at high concentrations to
accelerate the traceless Staudinger ligation reaction.[11] One
may also consider the addition of substituents to phenyl
groups of phosphinothioesters to retard the hydrolysis of the
P-N ylide to reduce the side Staudinger reduction product.[19]
Figure 6. Synthesis of H3K4su and its application as a probe of Sirt5
desuccinylation activity. A) Using dPPMT-NB-Su to convert H3K4AznL
into H3K4su. Reaction conditions and analysis were as same as for
the synthesis of Ub-K48su. B) Tandem MS analysis of the trypsinized
Ksu-containing fragment of the 48-h reaction and UV-treated product
of H3K4AznL. C) Desuccinylation of the H3K4su-H4 tetramer by Sirt1
and Sirt5. 180 mm H3K4su-H3 tetramer was incubated with or without
0.5 mm Sirt1/Sirt5 in the presence of 10 mm NAD+ for 4 h. After
reactions, proteins were analyzed by western blot analysis using anti-
H3 and pan anti-Ksu antibodies.
Acknowledgements
Support of this work was provided from National Institute of
Health (grants R01CA161158, R01GM098596, and
R01GM121584) and Welch Foundation (grant A-1715).
H3K4AznL was successfully converted to H3K4su, which
was detected at high levels by the pan anti-Ksu antibody. The
control reaction with wild type H3 led to a very low level of
lysine succinylation. To confirm K4 succinylation, the 48-h
incubation and UV-treated product of H3K4AznL was
trypsinized and analyzed by the MS/MS. The tandem MS
spectrum of the Ksu-containing fragment clearly showed
succinylation at K4 (Figure 6B). After confirming the succi-
nylation site, we directly used this 48-h incubation and UV-
treated product of H3K4AznL to assemble an H3K4su-H4
tetramer that was subsequently used as a substrate to test
Sirt5 activity. As shown in Figure 6C, when Sirt5 and its
cofactor NAD+ were provided to the H3K4su-H4 solution,
the succinylation was actively removed from H3. On the
contrary, a control reaction with Sirt1 showed no succinyla-
tion removal from H3. These combined results unequivocally
approve show that Sirt5 but not Sirt1 is a histone desucciny-
lase.
Conflict of interest
The authors declare no conflict of interest.
Keywords: amber suppression · azidonorleucine ·
lysine acylation · protein modification ·
traceless Staudinger ligation
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4
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Manuscript received: November 21, 2016
Revised: December 13, 2016
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Protein Modifications
Z. A. Wang, Y. Kurra, X. Wang, Y. Zeng,
Y.-J. Lee, V. Sharma, H. Lin, S. Y. Dai,*
W. R. Liu*
&&&& — &&&&
A Versatile Approach for Site-Specific
Lysine Acylation in Proteins
Azidonorleucine, an azide-containing
amino acid, is genetically encoded and
incorporated into model proteins. This
incorporation followed by traceless Stau-
dinger ligation potentiates the synthesis
of proteins with a myriad of site-specific
lysine acylations.
6
www.angewandte.org
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!