A Genetically Encoded Allysine for the Synthesis of Proteins with Site-Specific Lysine Dimethylation

Article abstract of DOI:10.1002/anie.201609452

Using the amber suppression approach, N^?-(4-azidobenzoxycarbonyl)-δ,?-dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site-specific lysine dimethylation. Using this approach, dimethyl-histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.

Full text of DOI:10.1002/anie.201609452

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International Edition: DOI: 10.1002/anie.201609452
German Edition: DOI: 10.1002/ange.201609452
Protein Modification
A Genetically Encoded Allysine for the Synthesis of Proteins with Site-
Specific Lysine Dimethylation
Zhipeng A. Wang+, Yu Zeng+, Yadagiri Kurra+, Xin Wang, Jeffery M. Tharp, Erol C. Vatansever,
Willie W. Hsu, Susie Dai, Xinqiang Fang,* and Wenshe R. Liu*
Abstract: Using the amber suppression approach, Ne-(4-
azidobenzoxycarbonyl)-d,e-dehydrolysine, an allysine precur-
sor is genetically encoded in E. coli. Its genetic incorporation
followed by two sequential biocompatible reactions allows
convenient synthesis of proteins with site-specific lysine
dimethylation. Using this approach, dimethyl-histone H3 and
p53 proteins have been synthesized and used to probe functions
of epigenetic enzymes including histone demethylase LSD1
and histone acetyltransferase Tip60. We confirmed that LSD1
is catalytically active toward H3K4me2 and H3K9me2 but
inert toward H3K36me2, and methylation at p53 K372 directly
activates Tip60 for its catalyzed acetylation at p53 K120.
heterogeneity of methylated proteins, which makes it difficult
to separate homogenous proteins with site-specific lysine
methylation. Alternatively, native chemical ligation and
expressed protein ligation, two generally applied chemical
methods can be used for the synthesis of proteins with three
lysine methylation types.[4] However, both methods suffer
from limitations such as the requirement of a cysteine for the
ligation process and the obstacle to install lysine methylation
in the middle of a protein. Several groups have developed
approaches that combine the amber suppression-based muta-
genesis approach and photo- and chemical-based cleavage for
the synthesis of proteins with lysine monomethylation. In
these approaches, protected Ne-methyl-lysines are genetically
incorporated into proteins and then deprotected by photo-
and chemical-based cleavage of the protection groups.[5]
However, a similar method has not been developed for the
synthesis of proteins with lysine dimethylation or lysine
trimethylation. Chin and co-workers previously described
a multi-step strategy for the synthesis of hi^stones with lysine
dimethylation that involves the genetic incorporation of
a protected lysine at a designated lysine site of a histone,
global protection of all other lysine residues and N-terminal
amine in the expressed histone, the removal of the protection
group from the genetic encoded modified lysine to recover
lysine at the designated site, reductive alkylation with
formaldehyde to install lysine dimethylation at the designated
site, and the final removal of the global protection group to
afford a dimethyl-histone.[6] Although elegant, this approach
cannot be applied to proteins that are sensitive to denaturing
conditions used for global protection and deprotection of
lysine residues and N-terminal amine. Its incompatibility with
cysteine that was not present in the original model histone is
also a concern.
P
rotein lysine methylation is a reversible posttranslational
modification that was originally discovered in histones but
occurs also in many non-histone proteins.[1] There are three
levels of lysine methylation, namely mono-, di-, and trime-
thylation that coordinate with other histone modifications to
regulate chromatin-based transcriptional control and shape
inheritable epigenetic programs in the eukaryotes.[2] Besides
its epigenetic roles of chromatin regulation, lysine methyl-
ation also serves critical functions in regulating activities of
transcription factors such as p53 and NF-kB.[3] Proteins with
site-specific lysine methylation can be potentially synthesized
by incubating target proteins with histone methyltransferases
(HMTs). However, not all lysine methylation sites have their
corresponding HMTs identified. In addition, the promiscuity
of HMTs and the three levels of methylation add to high
[*] Z. A. Wang,[+] Dr. Y. Zeng,[+] Dr. Y. Kurra,[+] J. M. Tharp,
E. C. Vatansever, W. W. Hsu, Prof. Dr. W. R. Liu
Department of Chemistry, Texas A&M University
Corner of Ross and Spence Streets, College Station, TX 77843 (USA)
E-mail: wliu@chem.tam.edu
In order to site-specifically install lysine dimethylation in
proteins, we envisioned that allysine (AlK, Figure 1A),
a naturally occurring derivative of lysine in elastin and
collagen,[7] can be genetically encoded using the amber
suppression mutagenesis approach and then undergo reduc-
tive amination with dimethylamine for the installation of site-
specific lysine dimethylation in proteins. Given the concern of
the cellular toxicity from its side chain aliphatic aldehyde,
AlK was not directly used. Instead a precursor amino acid, Ne-
(4-azidobenzoxycarbonyl)-d,e-dehydrolysine (AcdK, Fig-
ure 1A) that shields the side chain aldehyde was designed.
AcdK has an azidobenzoxycarbonyl moiety whose reduction
with a phosphine will trigger a self-cleavage process to release
d,e-dehydrolysine.[8] d,e-Dehydrolysine does not stably exist
in water and hydrolyzes instantaneously to form AlK. By
genetically incorporating AcdK into proteins followed by
Dr. X. Wang, Prof. Dr. S. Dai
Department of Plant Pathology and Microbiology, Institute for Plant
Genomics, Office of the Taxes State Chemist, Department of
Veterinary Pathobiology
College Station, TX 77843 (USA)
Prof. Dr. X. Fang
Fujian Institute of Research on the Structure of Matter, Chinese
Academy of Science, Fuzhou
Fujian 350002 (P.R. China)
E-mail: xqfang@fjirsm.ac.cn
[+] These authors contributed equally to this work.
Supporting information (synthesis and characterization of AcdK,
selection of AcdKRS, expression of proteins and their treatment to
install mono- and dimethylation, and assay conditions of LSD1 and
Tip60) and the ORCID identification number(s) for the author(s) of
this article can be found under http://dx.doi.org/10.1002/anie.
201609452.
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Figure 1. Application and synthesis of AcdK: A) Structures of AlK and AcdK. B) A diagram that illustrates the genetic incorporation of AcdK
followed by Staudinger reduction, self-cleavage of the para-aminobenzyloxycarbonyl group, enamine hydrolysis, and reductive amination to
generate a dimethyllysine in a protein. C) The synthesis of AcdK. It starts with l-glutamate and finishes with a dilithium salt of AcdK.
Staudinger reduction with tris-(2-carboxyethyl)phosphine
(TCEP) to recover AlK and then reductive amination with
dimethyllysine in the presence of NaCNBH3, proteins with
site-specific lysine dimethylation can be potentially synthe-
sized (Figure 1B). Since both Staudinger reduction and
reductive amination can be carried out in mild physiological
conditions, this approach can be generally applied for the
synthesis of proteins with site-specific lysine dimethylation as
well as the synthesis of proteins with site-specific lysine
monomethylation by simply changing dimethylamine to
methylamine in the reductive amination step. A synthetic
route of AcdK shown in Figure 1C that starts with l-
glutamate and finishes as dilithium salt of AcdK was designed
and successfully tested. Although the overall synthesis
involves 9 steps, gram quantities of AcdK have been routinely
produced.
In order to use amber codon to genetically encode AcdK
in E. coli, we employed an engineered pyrrolysyl-tRNA
synthetase (PylRS)-tRNAPyl system.[9] Wild-type and mutant
PylRS-tRNAPyl pairs have been widely used for the genetic
incorporation of a large number of lysine and phenylalanine
derivatives into proteins at amber mutation sites in a series of
cell strains.[10] A PylRS gene library in which codons for four
active site residues, Y306, L309, C348, and Y384 of PylRS
were randomized was first constructed. A broadly adopted
and double-sieved selection protocol[10a] was applied for the
selection of PylRS mutants that charge tRNAPyl with AcdK.
The mutant, together with AcdK and tRNAPyl, that displays
the best amber suppression efficiency in E. coli has mutations
as L309T/C348G/Y384F and is coined as AcdKRS (Support-
superfolder green fluorescent protein (sfGFP) with an
amber mutation at D134 and a C-terminal 6 ꢀ His tag were
used to cotransform E. coli BL21(DE3) cells. Growing the
transformed cells in the presence of AcdK afforded full-
length sfGFP (sfGFP-D134AcdK), which was markedly
contrasted with non-detectable full-length sfGFP expressed
in the absence of AcdK (Figure 2A). In the presence of
0.5 mm AcdK, 7 mgLÀ1 sfGFP-D134AcdK was expressed.
This observation demonstrates the high selectivity of
AcdKRS toward AcdK. The electrospray ionization mass
spectrometry (ESI-MS) analysis of the purified sfGFP-
D134AcdK displayed two major peaks (Figure 2B). One
major peak at 28013 Da agrees well with the theoretical
molecular weight of sfGFP-D134AcdK (28013 Da). The
other major peak at 27839 Da implies partial reduction of
AcdK in sfGFP-D134AcdK to AlK (the theoretic mass of
sfGFP-D134AlK is 27839 Da). This was confirmed via label-
ing the purified sfGFP-D134AcdK readily with a hydroxyl-
amine-conjugating fluorescein dye (SI, Figure S2; the original
sfGFP fluorescence was quenched by denaturing the protein
at the boiling temperature). Since no reducing reagents were
provided during the purification process, this partial reduc-
tion is possibly due to reactions with endogenous thiol-
containing reagents such as glutathione and H2S in E. coli
cells.[11] Given that AcdK is used as a precursor to install AlK
in a protein, this partial conversion of AcdK to AlK is not
a concern but a benefit. The purified sfGFP-D134AcdK was
later converted quantitatively to sfGFP-D134AlK by reacting
with TCEP (Figure 2C). The minor peak at 27857 Da in
Figure 2C represents the hydrated form of sfGFP-D134AlK
(the theoretical mass: 27857 Da). Partial hydration of an
aliphatic aldehyde in water is a general observation. Given its
reversible nature, this hydration is not expected to interfere
with the following reductive amination. SfGFP-D134AlK was
ing Information (SI), Figure S1).
A plasmid pEVOL-
AcdKRS that contains genes encoding AcdKRS and tRNAPyl
was then constructed. This plasmid and another plasmid
pBAD-sfGFP-D134TAG that contains a gene encoding
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Figure 2. Genetic incorporation of AcdK into a model protein sfGFP for the lysine di- and monomethylation installation: A) The site-specific
incorporation of AcdK into sfGFP at its D134 position to produce sfGFP-D134AcdK. Cells were transformed with two plasmids containing genes
for AcdKRS, tRNAPyl, and sfGFP with an amber mutation at its D134 position and grown in LB media supplemented with or without 0.5 mm AcdK.
B,C) The ESI-MS spectra of purified sfGFP-S2AcdK (B) and its TCEP-reduced product (C). D) The selective installation of Kme2 at the D134
position of sfGFP. SfGFP-D134AcdK was reduced with 5 mm TCEP in PBS buffer at pH 7 for 2 h followed by the reaction with 100 mm
dimethylamine and 5 mm NaCNBH3 in PBS buffer at pH 7 for 8 h for the installation of Kme2 to form sfGFP-D134Kme2. The original sfGFP-
D134AcdK was used as a control in the Western blot and SDS-PAGE analysis. The top panel shows a Western blot probed by anti-Kme2 and the
bottom panel shows a Coomassie blue stained gel for the two proteins. E,F) The ESI-MS spectra of sfGFP-D134Kme2 (E) and sfGFP-D134Kme
(F).
subsequently converted to sfGFP with dimethyllysine (Kme2)
installed at D134 (sfGFP-D134Kme2) by incubating with
dimethylamine and NaCNBH3. The installation of Kme2 was
confirmed by both Western blot and ESI-MS analyses (Fig-
ure 2D,E; the theoretic molecular weight of sfGFP-
D134Kme2 is 27868 Da). The Kme2 site was further con-
firmed with the tandem MS (MS/MS) analysis of trypsinized
sfGFP-D134Km2 fragments (SI, Figure S3). We also tested
the synthesis of sfGFP with methyllysine (Kme) installed at
D134 (sfGFP-D134Kme) by reacting sfGFP-D134AlK with
methylamine and NaCNBH3. The finally synthesized sfGFP-
D134Kme displayed an ESI-MS peak at 27855 Da that agrees
well with the theoretical mass (27854 Da) (Figure 2F). The
formation of Kme in sfGFP-D134Kme cannot be independ-
ently confirmed with the Western blot analysis due to the lack
of a commercial pan anti-Kme antibody. For both Staudinger
reduction and reductive amination, reactions were carried out
in the PBS buffer at pH 7 and room temperature, no protein
aggregation or quenching of the sfGFP fluorescence was
observed, indicating that both reactions are compatible with
preserving proteins in their native forms.
and used them to probe substrate specificities of LSD1. LSD1
is a FAD-dependent histone demethylase that selectively
targets H3 at K4 and K9 positions to remove their mono- and
dimethylation but is inert toward other methylated H3
lysines.[12] In order to synthesize H3K4me2 and H3K9me2,
two precursor proteins H3K4AcdK and H3K9AcdK were
expressed and purified similarly as sfGFP-D134AcdK (Fig-
ure 3A). H3K36AcdK was synthesized in parallel as a control.
All three proteins were then processed with Staudinger
reduction and reductive amination to make H3K4me2,
H3K9me2, and H3K36me2, respectively. The formation of
the three dimethyl-H3 variants was confirmed with their
detection by the pan anti-Kme2 antibody in the Western blot
analysis (Figure 3B) and the Kme2 installation sites were
further confirmed with the tandem MS analysis of trypsinized
fragments of three histone proteins (Figure 3C–E). The
installation of Kme2 at H3K4 was also independently
confirmed with Western blotting by anti-H3K4me2 (SI,
Figure S5). We also proceeded to synthesize H3K4me and
H3K9me. However, commercial pan anti-Kme2/Kme anti-
bodies from both Abcam and PTM Biolabs failed to
recognize them although they detected dimethyl-H3 variants
successfully (SI, Figure S6). The three synthesized dimethyl-
H3 variants were then used as substrates of LSD1 to test its
After the demonstration of the designed method for the
synthesis of sfGFP installed site-specifically with Kme2, we
next moved to synthesize three dimethyl-histone H3 variants
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Figure 3. Synthesis of H3K4me2, H4K9me2, and H3K36me2: A) SDS-PAGE and Western blot analyses of purified AcdK-containing H3 variants.
Two plasmids pEVOL-AcdKRS and petDeut-1-H3 with a gene coding H3 with an amber mutation were used to co-transform E. coli BL21(DE3)
cells. The transformed cells were grown in LB medium to OD 0.8. H3 expression was induced with the addition of 0.2% arabinose, 1 mm IPTG,
and 2 mm AcdK. The expressed H3 variants were purified using Ni-NTA resins. B) The Western blotting analysis of AcdK-containing H3 variants
after their Staudinger reduction with TCEP and reductive amination with dimethylamine. Reaction conditions are the same as shown in Figure 2D.
C–E) Tandem mass spectrometry analysis of Kme2-containing and trypsinized fragments of H3K4me2, H3K9me2, and H3K36me2.
demethylation activities. As expected, LSD1 actively
removed dimethylation at K4 and K9 but was inert toward
dimethylation at K36 of H3 (Figure 4A). To show that the
designed method can be applied to large proteins with
multiple cysteine residues, we chose to work on p53. P53 is
a tumor antigen and a transcription factor with 393 amino
acids. Its mutations are associated with 60% of tumors. P53
undergoes methylation at K372 that activates its transcription
activity.[13] It has been speculated that this activating role is
achieved through the recruitment of Tip60, a histone acetyl-
transferase that has a methyllysine-binding chromodomain
and acetylates p53 at K120.[14] We determined to confirm this
by using p53 proteins that are site-specifically installed with
lysine mono- and dimethylation. P53 with AcdK incorporated
at K372 was first expressed in E. coli similarly as sfGFP-
D134AcdK (SI, Figure S7). P53 typically has very low
expression yields in E. coli.[15] Therefore sfGFP with a C-
terminal 6 ꢀ His tag was fused to the C-terminus of p53 that
also had a N-terminal GST tag for boosting up its expression
in E. coli and easy separation from cell lysate. The expressed
protein then underwent Staudinger reduction and reductive
amination to generate p53 with mono- and dimethylation at
K372 (p53-K372me and p53-K372me2), respectively. As
shown in Figure 4B, the formation of p53-K372me and p53-
K372me2 was confirmed with the Western blot analysis using
anti-p53-K372me and pan anti-Kme2 antibodies. A wild-type
p53 was also expressed as a control. All three proteins were
then reacted with Tip60 in the presence of acetyl-CoA and
subsequently probed by an anti-p53-K120ac antibody. As
Figure 4. Probing functions of epigenetic enzymes with synthetic
dimethyl-H3 and p53 proteins: A) LSD1 activities on three dimethyl-H3
variants. 1 mg H3K4me2, H3K9me2, and H3K36me2 were incubated
with 0.1 mg for 8 h before they were analyzed in SDS-PAGE and probed
by anti-Kme2 and anti-H3 antibodies in Western blotting. B) Methyl-
ation at p53 K372 activates Tip60-catalyzed acetylation at p53 K120.
0.1 mg wild-type (wt) p53, p53-K372me, and p53-K372me2 was incu-
bated with 0.01 mg Tip60 for 2 h before they were analyzed by SDS-
PAGE and probed with antibodies such as anti-p53-K372me, anti-
Kme2, and anti-p53-K120ac in Western blotting. C) Quantified activa-
tion effects of p53 K372 methylation on p53 K120 acetylation by Tip60.
shown in the bottom panel in Figure 4B, Tip60 catalyzed
K120 acetylation of all three proteins however K372 meth-
ylation significantly enhanced the reaction. Both mono- and
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dimethylation at K372 improved K120 acetylation by 2.5 to 3
folds (Figure 4C). This is the first evidence to show that K372
methylation of p53 directly recruits Tip60 to p53 for its
acetylation at K120.
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In summary, a method that allows expedient synthesis of
proteins with site-specific lysine dimethylation has been
successfully demonstrated. The method combines the amber
suppression-based mutagenesis with two biocompatible
organic reactions to install site-specific lysine dimethylation
in proteins. Given its specificity, biocompatibility, and sim-
plicity, it can be generally applied to synthesize proteins with
site-specific lysine dimethylation for their functional inves-
tigation. Besides proteins with lysine dimethylation, we
showed that the same method could be applied to synthesize
proteins with lysine monomethylation. Other posttransla-
tional lysine alkylation types that can be potentially installed
site-specifically into proteins using the presented method
include the enzymatic deoxyhypusine and hypusine formation
in eukaryotic eIF5A and metabolic lysine glycation subtypes
such as carboxymethylation.[16] Given that allysine itself
naturally occurs in elastin and collagen for their crosslink-
ing,[7] the application of our currently reported method in
studying the elastin and collagen fibril formation is also
anticipated.
Acknowledgements
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Support of this work was provided from the National
Institutes of Health (grant CA161158), National Science
Foundation (grant CHE-1148684), Welch Foundation (grant
A-1715), and the National Natural Science Fundation of
China (grants 21402199 and 21502192). We thank Dr.
Yohannes H. Reznom at the Laboratory for Biology Mass
Spectrometry and Dr. Larry Dangott at the Protein Chemis-
try Lab of Texas A&M University for characterizing our
proteins with mass spectrometry.
Keywords: allysine · amber suppression · dimethyllysine ·
genetic code expansion · lysine dimethylation
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Received: September 26, 2016
Published online: && &&, &&&&
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Communications
Protein Modification
Z. A. Wang, Y. Zeng, Y. Kurra, X. Wang,
J. M. Tharp, E. C. Vatansever, W. W. Hsu,
S. Dai, X. Fang,*
W. R. Liu*
&&&& — &&&&
A Genetically Encoded Allysine for the
Synthesis of Proteins with Site-Specific
Lysine Dimethylation
Expedient protein synthesis: An allysine
precursor, Ne-(4-azidobenzoxycarbonyl)-
d, e-dehydrolysine is genetically encoded
in E. coli. Its incorporation followed by
Staudinger reduction and reductive ami-
nation allows the synthesis of proteins
with site-specific lysine dimethylation.
6
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Angew. Chem. Int. Ed. 2016, 55, 1 – 6
These are not the final page numbers!