Labeling lysine acetyltransferase substrates with engineered enzymes and functionalized cofactor surrogates

Article abstract of DOI:10.1021/ja311636b

Elucidating biological and pathological functions of protein lysine acetyltransferases (KATs) greatly depends on the knowledge of the dynamic and spatial localization of their enzymatic targets in the cellular proteome. We report the design and application of chemical probes for facile labeling and detection of substrates of the three major human KAT enzymes. In this approach, we create engineered KATs in junction with synthetic Ac-CoA surrogates to effectively label KAT substrates even in the presence of competitive nascent cofactor acetyl-CoA. The functionalized and transferable acyl moiety of the Ac-CoA analogs further allowed the labeled substrates to be probed with alkynyl or azido-tagged fluorescent reporters by the copper-catalyzed azide-alkyne cycloaddition. The synthetic cofactors, in combination with either native or rationally engineered KAT enzymes, provide a versatile chemical biology strategy to label and profile cellular targets of KATs at the proteomic level.

Full text of DOI:10.1021/ja311636b

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Communication
Labeling Lysine Acetyltransferase Substrates with
Engineered Enzymes and Functionalized Cofactor Surrogates
Chao Yang, Jiaqi Mi, You Feng, Liza Ngo, Tielong Gao, Leilei Yan, and Yujun George Zheng
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja311636b • Publication Date (Web): 09 May 2013
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Labeling Lysine Acetyltransferase Substrates with Engineered Enzymes and Func-
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Chao Yang, Jiaqi Mi, You Feng, Liza Ngo, Tielong Gao, Leilei Yan, Yujun George Zheng*
Department of Chemistry and The Centre for Biotechnology and Drug Design, Georgia State University, PO BOX 4098,
Atlanta, GA 30302-4098
Supporting Information Placeholder
Enzyme
S
ubstrate Labelin
g
ABSTRACT: Elucidating biological and pathological functions
of protein lysine acetyltransferases (KATs) greatly depends on the
knowledge of the dynamic and spatial localization of their enzy-
matic targets in the cellular proteome. Here we report the design
and application of chemical probes for facile labeling and detec-
tion of substrates of the three major human KAT enzymes. In this
approach, we attempted to create engineered KATs in junction
with synthetic Ac-CoA surrogates to effectively label KAT sub-
strates even in the presence of competitive nascent cofactor ace-
tyl-CoA. The functionalized and transferable acyl moiety of the
Ac-CoA analogs further allowed the labeled substrates to be
probed with alkynyl or azido-tagged fluorescent reporters by the
copper-catalyzed azide−alkyne cycloaddition. The synthetic co-
factors, in combination with either native or rationally engineered
KAT enzymes, provide a versatile chemical biology strategy to
label and profile cellular targets of KATs at the proteomic level.
Engineering
HS CoA
H
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wt-KAT
mut-KAT
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Protein
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or N3
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Cofactor
Design
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CoA
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m = 2
3
,
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H
N
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nN
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CH
N
4
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3
n = 2, 3
O
n
Substrate Visualization
Figure 1. Acetyl-CoA analogs combined with a protein-
engineering approach to label KAT substrates.
Elucidation of the molecular targets of KATs is a key step to-
ward fully dissecting the epigenetic roles of KATs in gene regula-
tion and their functions beyond the chromatin biology realm.
Dynamic lysine acetylation of proteins is involved in a variety
of fundamental biological processes including epigenetic pro-
graming, cell cycle, apoptosis, metabolism, and signal transduc-
tion . Acetylation is introduced by protein lysine acetyltransferas-
es (KATs), also oft-referred to as histone acetyltransferases
Mass spectrometry (MS)-based technologies have provided a
1
1e,3a,3b
great deal of information about acetylated proteins
, albeit
with little on enzyme-substrate correlations. Protein microarray is
another appealing approach in KAT substrate identification .
(HATs) and protein acetyltransferases (PATs), which transfer the
3c
acetyl group from the co-substrate acetyl-coenzyme A (acetyl-
CoA, Ac-CoA) to the epsilon-amino group of specific lysine resi-
dues in proteins. Over the past decade, several KAT members
have been identified and characterized both genetically and bio-
chemically. Based on the sequence and structural similarities,
KAT enzymes are classified into several major families, including
Recently, synthetic Ac-CoA analogs have been explored to identi-
fy KAT substrates, which provides a great chemical biology strat-
egy to interrogate the acetylome of particular KATs . In line with
5
this chemical biology paradigm, we attempted to create engi-
neered KATs in junction with synthetic Ac-CoA surrogates to
establish bioorthogonal probes to investigate cellular substrates of
KAT enzymes. A panel of Ac-CoA analogs with alkynyl or azido
functional groups were synthesized as cofactor substitutes for
selective labeling of KAT substrates. Meanwhile, the active site of
the key KATs was engineered in order to expand the cofactor
binding capability of the enzymes to accommodate the bulkier
synthetic cofactors (Fig. 1). By screening the activities of KAT
enzymes to individual Ac-CoA analog, several enzyme-cofactor
pairs were identified and applied to the investigation of cellular
substrates of KATs.
A set of Ac-CoA analogs was synthesized to provide potential
active cofactors for the engineered KATs (Supplementary Table
S1). These compounds include 4-pentynoyl CoA (4PY-CoA), 5-
hexynoyl CoA (5HY-CoA), 6-heptynoyl CoA (6HY-CoA), 3-
azidopropanoyl CoA (3AZ-CoA) and 4-azidobutanoyl CoA
(4AZ-CoA). The chemical coupling of carboxylic acid with HS-
CoA was achieved with N,N'-dicyclohexylcarbodiimide (DCC)
2
the GCN5/PCAF family, the MYST family, and the p300/CBP .
Recent biochemical and proteomic studies have revealed the
existence of hundreds to thousands of acetylated proteins
throughout the cell, which suggests that acetylation takes part in
1
b,1e,1f,3
nearly every facet of cell physiology
. Furthermore, signifi-
cant amounts of evidence point toward that altered KAT expres-
sion and activity are characteristic to inflammation, diabetes, can-
4
cer, neurological disorders, and many other diseases . While the
importance of KATs in physiology and disease is well recognized,
functional annotation of KAT enzymes in regulating key biologi-
cal pathways is rather understudied. Especially, how the acety-
lome of individual KAT enzymes distinguishes from one another
and how the substrate distribution of KATs is affected by differ-
ent cellular contexts remain to be clarified. A clear biochemical
and structural understanding of KAT substrate specificity and the
impact of lysine acetylation in (patho)physiology is in great de-
mand.
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with slight modification from the literature procedure (Supple-
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mentary Fig. S1). We noted that 4PY-CoA was previously uti-
ic analogs rather than the nascent cofactor, several double-point
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lized as a cofactor for p300 catalysis . The installed alkynyl and
azido functional groups will facilitate subsequent detection and
characterization of labeled KAT substrates.
mutants were generated by combining the most active single mu-
tants (T612G, L531A, L531G, I576A, and F622A). As shown in
Fig. 2b, most of these double mutants retained certain activities
toward the analogs. Especially, GCN5-T612G/F622A and -
T612G/L531A mutants exhibited excellent activity toward 5HY-
CoA. MALDI-MS analysis of the reaction mixtures further con-
firmed that H3-20 peptide can be efficiently modified by GCN5-
T612G/F622A and -T612G/L531A with 5HY-CoA (Fig. 2c, 2d,
and additional MS data in Fig. S5). These data highlight that
GCN5-T612G/F622A and -T612G/L531A in paired with 5HY-
CoA are excellent enzyme-cofactor pairs for selective chemical
labeling of GCN5 substrates.
The GCN5/PCAF KATs are key players in biological acetyla-
tion. Their structures have been well characterized which provides
2a,7
atomic details of the Ac-CoA binding site . Examination of the
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crystal structure of hGCN5─Ac-CoA complex (PDB 1Z4R, ref. )
revealed several conserved bulky residues in the Ac-CoA binding
pocket of the enzyme, i.e. L531, M534, I576, F578, T612, F622,
and Y645 (Fig. 2a). To expand the cofactor binding pocket so that
it can tolerate larger size acyl groups, we replaced each of these
residues with smaller amino acid residues, i.e. alanine or glycine.
Furthermore, based on the initial enzymatic screening data, sever-
al double-point mutants were generated to further boost the acyl-
transferring activity of the engineered GCN5.
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The MYST proteins represent another major KAT family in the
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higher organisms . To create chemical probes for identifying
substrates of the MYST family, we engineered the active site of
the MYST member MOF in a similar manner as described above
for GCN5. In the crystal structure of MOF─Ac-CoA complex
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(PDB 2Y0M, ref. ), the acetyl moiety of the cofactor is encircled
by bulky residues V314, I317, I333, P349, P352 and L353 (Fig.
3a). H273 is another potential residue that may affect the enzyme
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activity . We performed mutation of each residue to Ala or Gly
to expand the enzyme active site for acyl-CoA binding.
Figure 2. Identification of enzyme-cofactor pairs for GCN5
substrate labeling. (a) The bulky catalytic residues in the active site of
8
GCN5 (PDB 1Z4R) . (b) Heat map of engineered GCN5 activities to
different Ac-CoA analogs (see also Supplementary Fig. S3f). (c) MALDI-
MS of H3-20 peptide modified by GCN5-T612G/F622A and 5HY-CoA
(d) MALDI-MS of H3-20 peptide modified by the GCN5-
T612G/L531A−5HY-CoA pair.
Figure 3. Identification of enzyme-cofactor pairs for MOF
substrate labeling. (a) Key residues surrounding the active site of
MOF (PDB 2Y0M) . (b) Heat map of engineered MOF activities to Ac-
CoA analogs (see also Supplementary Fig. S4e). (c) MALDI-MS of H4-20
peptide modified by MOF-I317A/H273A−5HY-CoA pair.
To identify the engineered enzyme forms that are active to the
Ac-CoA substitutes, the entire panel of Ac-CoA analogs was
screened in histone modification reactions catalyzed by both the
wild-type (WT) and engineered GCN5 proteins. Typically, each
GCN5 was incubated with the N-terminal 20-aa H3 peptide (H3-
11
Activities of each MOF mutant toward the synthetic Ac-CoA
analogs were screened through enzymatic modification of the
histone H4 tail peptide containing the first 20 aa residues (H4-20)
by using the same fluorescent assay as described for GCN5 (Sup-
plementary Fig. S4). A heat map of MOF activity with respect to
individual Ac-CoA analogs was generated and shown in Fig. 3b.
Among the tested MOF mutants, H273A and H273G recognized
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0) and individual analogs. After the reaction, the mixtures were
treated with 7-diethylamino-3-(4'-Maleimidylphenyl)-4-
Methylcoumarin (CPM), a fluorogenic probe that becomes fluo-
9
rescent upon reacting with the sulfhydryl group in HS-CoA . The
fluorescence screening data were quantitated (Supplementary
Fig. S3) and presented in the heat map format in Fig. 2b. As ex-
pected, WT-GCN5 showed a strong activity toward Ac-CoA, the
cognate cofactor of KATs, but was inert toward all the Ac-CoA
substitutes. On the other hand, several engineered GCN5, e.g.
GCN5-L531A/G, -I576A, -T612A/G, and -F622A, exhibited ap-
preciable activities to the synthetic analogs at varied degrees. In
particular, the single mutant GCN5-T612G was active toward all
the analogs with 4AZ-CoA being the weakest. To further improve
the activity and selectivity of the engineered GCN5 to the synthet-
5
HY-CoA, and V314G recognized 6HY-CoA and 3AZ-CoA.
More strikingly, MOF-I317A was active toward all the Ac-CoA
analogs. In order to further improve the selectivity and activity of
MOF to the Ac-CoA analogs, several double-point mutants were
produced based on MOF-I317A which had a marked activity to
the analogs. The enzymatic assays revealed that, compared to the
single mutant MOF-I317A, the activity of all the double mutants
toward Ac-CoA and the analogs decreased by different degrees
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(
Fig. 3b). This likely suggests that MOF could not tolerate more
combinant H3 protein was incubated with each analog in the pres-
ence of GCN5-WT, -T612G, -T612G/F622A or -T612G/L531A.
Next, the mixture was probed with azide- or alkyne-coupled tet-
ramethylrhodamine (TAMRA) dyes with the CuAAC reaction.
The reaction mixture was resolved on SDS-PAGE and the gel was
scanned for fluorescence imaging. As shown in Fig. 4a, no label-
ing was observed in the reactions catalyzed by WT-GCN5, which
further confirmed that WT-GCN5 was inert to the synthetic ana-
logs. The three GCN5 mutants T612G, T612G/F622A, and
T612G/L531A, showed particularly strong labeling activity when
paired with 5HY-CoA. GCN5-T612G also showed activity to-
ward several other analogs such as 4PY-CoA, 6HY-CoA, and
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drastic mutations in its active site. We noted that MOF-
I317A/H273A retained about 6% activity to 5HY-CoA (Fig. 3b).
The activities of MOF-I317A and -I317A/H273A in taking the
Ac-CoA analogs were further corroborated in the MS experiments
(Fig. 3c, and Fig. S6).
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AZ-CoA, which is consistent with the screening results in the
heat map (Fig. 2b). In the labeling reactions, strong fluorescence
was only observed when H3 was subjected to the enzyme-
catalyzed modification, but barely observed in the enzyme-
negative controls, which excludes the possibility of nonspecific
reactions. These results again support that both GCN5-
T612G/F622A5HY-CoA and T612G/L531A5HY-CoA are
excellent enzyme-cofactor pairs for labeling of GCN5/PCAF sub-
strates.
To probe protein acetylation catalyzed by the MYST KATs,
both single-point mutant MOF-I317A and double-point mutant
MOF-I317A/H273A were applied to label recombinant H4 in the
presence of the synthetic Ac-CoA analogs. Again, following the
enzymatic reaction, CuAAC chemistry was applied for fluores-
cent detection. As shown in Fig. 4a, MOF-I317A and -
I317A/H273A showed a clear histone labeling activity when
5HY-CoA was used as the acyl donor. These encouraging results
demonstrate that both MOF-I317A and -I317A/H273A in combi-
nation with 5HY-CoA are suitable enzyme-cofactor matches to
label substrates of the MYST KATs. 3AZ-CoA was also a very
effective cofactor for labeling by MOF-I317A.
Having validated the activities of the engineered GCN5 and
MOF in conjugation with the synthetic cofactors on their cognate
histone substrates, we attempted to profile the proteome-wide
targets of KATs from the cellular contexts. As a principle of con-
cept, we performed fluorescent labeling of KAT substrates from
human embryonic kidney 293T cells. The cells were lysed in the
®
M-PER buffer, treated with the KAT-cofactor pairs, then labeled
with fluorescent reporters via the CuAAC and visualized on SDS-
PAGE. Multiple modified protein bands were readily visualized
on in-gel fluorescence of the cell lysate treated with GCN5-
T612G/L531A−5HY-CoA and T612G/F622A−5HY-CoA pairs
(lane 5 and 8 in Fig. 4b). The addition of Ac-CoA suppressed the
fluorescent labeling, which was in good agreement with the com-
petitive nature of 5HY-CoA with respect to Ac-CoA (lane 6 and 9
in Fig. 4b). The lack of fluorescence in WT-GCN5−5HY-CoA
pairing (lane 2 in Fig. 4b) as well as in the enzyme-negative con-
trol (lane 3 in Fig. 4b) further supports the matching characteristic
of the engineered KAT-cofactor pairs and thereby endogenous
GCN5 in the 293T cells did not interfere with the assay.
We also performed fluorescent labeling of protein substrates of
the MYST KATs with the engineered MOF and cofactor substi-
tutes. Both MOF-I317A and -I317A/H273A, which showed ap-
preciable activity in histone modification assays, were used in
conjugation with selected Ac-CoA analogs for target labeling. On
the fluorescent image of the SDS-PAGE gel, multiple labeled
lanes were observed when the cell lysate was treated with MOF-
I317A in the presence of the synthetic analogs (lane 13, 16, 19,
Figure 4. In-gel fluorescent detection of KAT substrates with
the bioorthogonal chemical probes. (a) Histone labeling by KATs
and Ac-CoA analogs. Commassie blue staining was shown in right panel.
The whole gel images were shown in Supplementary Fig. S8. (b) Cell
lysate labeling by KATs and selected Ac-CoA analogs. The lower panel
shows the commassie blue staining.
2
3, 27 in Fig. 4b). Consistent with the results of the histone modi-
The clickable feature of the alkyne- or azido-containing Ac-
CoA analogs provides a unique advantage for selective labeling,
visualization, and further characterization of KAT targets by using
the copper-catalyzed azide−alkyne cycloaddition (CuAAC) chem-
fication assays, MOF-I317A showed labeling activity toward the
tested analogs, with 5HY-CoA and 3AZ-CoA being particularly
strong. As expected, the double-point mutant MOF-I317A/H273A
effectively labeled the cell lysate with 5HY-CoA (lane 31 in Fig.
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istry (Fig. 1) . First, we performed fluorescent detection of H3
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b)
protein modification mediated by the engineered GCN5. The re-
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The other important KAT family in the mammalian system is
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p300/CBP. So far a clear p300/Ac-CoA complex structure is not
yet available, which makes it difficult to determine what residues
should be selected for active site engineering. Given this limita-
tion, we tested the histone H4 peptide labeling activity of p300
HAT domain protein with each synthetic Ac-CoA analog. Strik-
ingly, in addition to the previously reported active cofactor 4PY-
Experimental details for chemical synthesis, DNA mutation and
protein expression, enzymatic assays, and additional data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Corresponding Author
To whom correspondence should be addressed: Y. G. Zheng.
Phone: (404) 413-5491. Email: yzheng@gsu.edu
5b,6
CoA , the azido-containing analog 3AZ-CoA was found to be
strongly recognized by p300 HAT domain (Supplementary Fig.
S7). This data suggests that 3AZ-CoA is another excellent Ac-
CoA surrogate to identify protein targets of the p300/CPB family
KATs. To validate this result, we examined p300 by pairing it
with the synthetic analogs for labeling the H4 protein. The recom-
binant H4 protein was mixed with different analogs and p300
HAT domain, followed by CuAAC with an alkyne/azide-
conjugated TAMRA dyes. As seen in Fig. 4a, most efficient la-
beling was observed in the p300─3AZ-CoA pair. As a control,
there was no modification for the enzyme-negative sample. Also,
p300 paired with the other analogs gave much weaker histone
labeling activity than the p300─3AZ-CoA pair. Replacing the
p300 HAT domain with full-length p300 yielded similar results
ACKNOWLEDGMENT
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This work was supported by AHA Grant 12GRNT12070056 and
NIH Grant R01GM086717.
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C.; Qian, J.; Zhao, Y.; Boeke, J. D.; Berger, S. L.; Zhu, H. Cell 2009, 136,
1
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celerated by developing innovative chemical biology probes . In
this study, we demonstrated the success of using rationally engi-
neered proteins in conjunction with synthetic Ac-CoA analogs, as
new chemical tools, to efficiently label KAT substrates. For the
GCN5/PCAF family, we found that both GCN5-
T612G/F622A5HY-CoA and GCN5-T612G/L531A5HY-
CoA are superior matching pairs for selective labeling of sub-
strates of GCN5/PCAF KATs. For the MYST family, we identi-
fied MOF-I317A and MOF-I317A/H273A that showed marked
activity with 5HY-CoA. MOF-I317A was also very active toward
1
073; (d) Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman,
M.; Walther, T. C.; Olsen, J. V.; Mann, M. Science 2009, 325, 834; (e)
Basu, A.; Rose, K. L.; Zhang, J.; Beavis, R. C.; Ueberheide, B.; Garcia, B.
A.; Chait, B.; Zhao, Y.; Hunt, D. F.; Segal, E.; Allis, C. D.; Hake, S. B.
Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 13785; (f) Kim, G. W.; Yang, X.
J. Trends Biochem. Sci. 2011, 36, 211.
(4) (a) Saha, R. N.; Pahan, K. Cell Death Differ 2006, 13, 539; (b)
Avvakumov, N.; Cote, J. Oncogene 2007, 26, 5395; (c) Iyer, A.; Fairlie,
D. P.; Brown, L. Immunol. Cell Biol. 2012, 90, 39.
3
AZ-CoA. For the KAT p300, in addition to 4PY-CoA, a previ-
ously identified cofactor for the enzyme, we identified 3AZ-CoA
as another efficient cofactor for chemical labeling of p300 sub-
strates. Advancing from these new findings, several further stud-
ies and improvement would be warranted. For instance, thus far a
clear biochemical and structural understanding of how different
KAT families showed varied abilities in recognizing the Ac-CoA
analogs is not available. A systematic kinetic study will quantita-
tively evaluate the efficiencies of the KATs (both wt- and mutant
forms) for the synthetic cofactors to assure competent significance
of the bioorthogonal probes relative to the endogenous counter-
part. Also, in the current version, the KAT chemical probes are
limited to labeling protein substrates in cell lysates. Creation of
cell-permeable Ac-CoA analogs could be valuable for direct inter-
rogation of intracellular acetylation. The use of functionalized
acetate may also be a practical approach . In conclusion, we
identified for each of the three major families of human KAT
proteins the optimal enzyme-cofactor pairs that can be applied to
investigate new acetylation substrates. The synthetic Ac-CoA
analog cofactors in conjunction with their matched KATs are
expected to be versatile probes to identify KAT targets from ho-
mogenized cellular and tissue specimens, thereby expanding our
chemical tool repertoires for functional annotation of the acety-
lome in higher organisms.
(
5) (a) Yu, M.; de Carvalho, L. P.; Sun, G.; Blanchard, J. S. J Am Chem
Soc 2006, 128, 15356; (b) Yang, Y. Y.; Ascano, J. M.; Hang, H. C. J. Am.
Chem. Soc. 2010, 132, 3640.
(6) Yang, Y. Y.; Grammel, M.; Hang, H. C. Bioorg. Med. Chem. Lett.
2011, 21, 4976.
(7) Hodawadekar, S. C.; Marmorstein, R. Oncogene 2007, 26, 5528.
(
8) Schuetz, A.; Bernstein, G.; Dong, A.; Antoshenko, T.; Wu, H.;
Loppnau, P.; Bochkarev, A.; Plotnikov, A. N. Proteins 2007, 68, 403.
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319.
(
(10) Utley, R. T.; Cote, J. Curr. Top. Microbiol. Immunol. 2003, 274, 203.
(11) Kadlec, J.; Hallacli, E.; Lipp, M.; Holz, H.; Sanchez-Weatherby, J.;
Cusack, S.; Akhtar, A. Nat. Struct. Mol. Biol. 2011, 18, 142.
(12) (a) Yuan, H.; Rossetto, D.; Mellert, H.; Dang, W.; Srinivasan, M.;
Johnson, J.; Hodawadekar, S.; Ding, E. C.; Speicher, K.; Abshiru, N.;
Perry, R.; Wu, J.; Yang, C.; Zheng, Y. G.; Speicher, D. W.; Thibault, P.;
Verreault, A.; Johnson, F. B.; Berger, S. L.; Sternglanz, R.; McMahon, S.
B.; Cote, J.; Marmorstein, R. Embo J. 2012, 31, 58; (b) Sun, B.; Guo, S.;
Tang, Q.; Li, C.; Zeng, R.; Xiong, Z.; Zhong, C.; Ding, J. Cell Res. 2011,
5
b
2
1, 1262; (c) Yang, C.; Wu, J.; Sinha, S. H.; Neveu, J. M.; Zheng, Y. G. J.
Biol. Chem. 2012, 287, 34917.
(13) Meldal, M.; Tornoe, C. W. Chem. Rev. 2008, 108, 2952.
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Enzyme
Fluorophore
HS CoA
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