Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for Selectively Profiling Ubiquitin-Binding Proteins

Article abstract of DOI:10.1002/anie.201611659

Biochemical studies of cellular processes involving polyubiquitin have gained increasing attention. More tools are needed to identify ubiquitin (Ub)-binding proteins. We report diazirine-based photoaffinity probes that can capture Ub-binding proteins in c

Full text of DOI:10.1002/anie.201611659

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International Edition: DOI: 10.1002/anie.201611659
German Edition: DOI: 10.1002/ange.201611659
Photoaffinity Labelling
Chemical Synthesis of Diubiquitin-Based Photoaffinity Probes for
Selectively Profiling Ubiquitin-Binding Proteins
Jun Liang, Lin Zhang, Xiang-Long Tan, Yun-Kun Qi, Shan Feng, Haiteng Deng, Yijing Yan,
Ji-Shen Zheng, Lei Liu, and Chang-Lin Tian*
[
7]
Abstract: Biochemical studies of cellular processes involving
polyubiquitin have gained increasing attention. More tools are
needed to identify ubiquitin (Ub)-binding proteins. We report
diazirine-based photoaffinity probes that can capture Ub-
binding proteins in cell lysates, and show that diazirines are
preferable to aryl azides as the photo-crosslinking group, since
they decrease non-selective capture. Photoaffinity probes
containing at least two Ub units were required to effectively
capture Ub-binding proteins. Different capture selectivity was
observed for probes containing diubiquitin moieties with
different types of linkages, thus indicating the potential to
develop linkage-dependent probes for selectively profiling Ub-
binding proteins under various cellular conditions.
nology have been used, which has led to the capture and
characterization of approximately 150 Ub-binding pro-
[
8,9]
teins.
For instance, Pickart et al. used a K48-linked Ub4
aryl-azide-based probe to identify a novel Ub-binding site in
[
10]
the proteasome. Given that there are different UBDs that
specifically recognize the eight different types of polyUb
chains, we reasoned that incorporating photoaffinity groups
into different Ub chains may enable the identification of
specific UBDs.
Herein, we report diazirine-based photoaffinity probes to
capture Ub-binding proteins. These probes are tailor-
designed proteins generated through chemical protein syn-
thesis. Our work was inspired by the recent success of using
photoaffinity probes to capture proteins bound to other types
of PTMs. We found that photoaffinity probes with at least
two Ub units are required to effectively capture Ub-binding
proteins, and that diazirine is a more effective photo-cross-
linking group than aryl azide for capturing Ub-binding
proteins. Interestingly, we observed distinct capturing perfor-
mance for probes containing different diUbs, thus suggesting
the need to develop linkage-dependent probes to examine or
monitor Ub-binding proteins.
We initially designed aryl-azide-based photoaffinity
probes containing one Ub (1), Lys48-linked diUb (2), and
Lys63-linked diUb (3; Figure 1A). Because the binding
between Ub and UBDs usually involves the Ile44 hydro-
[
11]
A
s one of the most important posttranslational modifica-
tions (PTMs) in eukaryotes, ubiquitination is involved in
[1]
a wide range of cellular processes. This modification exhibits
higher complexity than other PTMs (e.g., phosphorylation)
because eight different types of polyubiquitin (polyUb)
chains are formed, through Met1, Lys6, Lys11, Lys27,
Lys29, Lys33, Lys48, or Lys63 linkages. Previous studies
have established that all eight polyUbs are present in cells and
[2]
[
3]
they show functional variations. For instance, Lys48-linked
polyUbs are signals for proteasomal degradation, whereas
Lys63-linked polyUbs are involved in signal transduction and
DNA repair. The distinct cellular signals of different polyUbs
are controlled by the recognition and decoding of polyUbs by
ubiquitin (Ub)-binding proteins containing ubiquitin-binding
[
12]
phobic patch of Ub, we put a photo-crosslinking phenyl
azide group at the Ala46 that neighbors Ile44. An affinity
handle (biotin) was placed at the C terminus of the probes to
enrich photo-crosslinked products. Probes containing both
isopeptide bonds and photo-crosslinking groups are difficult
to prepare through either expression or enzymatic constitu-
[
4]
domains (UBDs).
The identification of Ub-binding proteins, especially those
that can selectively recognize different polyUb linkages, is
fundamental to studying the cellular roles and regulatory
mechanisms of ubiquitination. For this purpose, bioinformat-
[
13]
tion. As a result, we carried out protein total synthesis,
[
5]
[6]
[14]
ics, yeast two-hybrid assays, and affinity pull-down tech-
which allows exquisite control at the atomic level.
For
example, to prepare 3, we divided it into three segments: 4, 5,
and 6 (Figure 2A). To construct the isopeptide bond,
a trifluoroacetic acid (TFA)-cleavable 1-(2,4-dimethoxy-
phenyl)-2-mercaptoethyl auxiliary (Aux) was introduced
[
*] J. Liang, Prof. Y. Yan, Prof. J.-S. Zheng, Prof. C.-L. Tian
Hefei National Laboratory for Physical Sciences at the Microscale
University of Science and University of Science and Technology of
China, Hefei 230026 (China)
[
15]
into 6 (see the Supporting Information).
Both Ala46
E-mail: cltian@ustc.edu.cn
residues from the two Ub units were mutated to Cys for
convenience of ligation, as well as the subsequent incorpo-
ration of photo-crosslinking groups.
X.-L. Tan, Y.-K. Qi, Prof. L. Liu
Department of Chemistry, Tsinghua University
Beijing 100084 (China)
Peptide segments 4–6 were synthesized using Fmoc-based
solid-phase peptide synthesis (SPPS, Fmoc = 9-fluorenylme-
thoxy-carbonyl) and purified using reversed-phase high-
performance liquid chromatography (RP-HPLC). Aux-
assisted ligation between 5 (1.2 equiv) and 6 (1.0 equiv) was
performed by using hydrazide-based native chemical ligation
L. Zhang, S. Feng, Prof. H. Deng
MOE Key Laboratory of Bioinformatics
Tsinghua-Peking Joint Center for Life Sciences
School of Life Sciences, Tsinghua University
Beijing 100084 (China)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201611659.
[16]
(NCL). This reaction was completed in approximately 12 h
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incubated on ice for 1.5 h and then exposed to 254 nm UV
light for the indicated period of time (1 or 5 min). After
streptavidin enrichment, the mixtures were analyzed by
sodium dodecyl sulfate-polyacrylamide polyacrylamide gel
electrophoresis (SDS-PAGE). Unexpectedly, 1, 2, and 3
crosslinked to tUIM with almost the same efficiency (Fig-
ure 3A). Furthermore, with an extended irradiation time
from 1 to 5 min, 2 and 3 crosslinked with two tUIM molecules
each, a result that is not consistent with a previous report
[20]
indicating that only one tUIM can bind K63-linked diUb.
To reconcile this discrepancy, we analyzed gel bands corre-
sponding to the crosslinked products by using liquid chroma-
tography coupled with tandem mass spectrometry (LC–MS/
MS). A crosslinked peptide fragment containing Cys121 of
tUIM was observed for all of the above experiments (Fig-
ure 3C), thus indicating that Cys121 might play an essential
[21]
role in all of the crosslinking products. A previous report
revealed that the nitrene intermediate produced after the loss
of N2 from an aryl azide tends to photoisomerize to
dehydroazepine, which can react with nucleophiles (Fig-
ure 3D). The observation that 2 and 3 crosslink to two tUIM
molecules through Cys indicates that aryl-azide-based cross-
linking reactions can occur through nucleophilic addition.
To further test the aryl-azide-based probes, we chose the
ubiquitin-associated 2 domain (UBA2) from the human
homologue of the yeast protein Rad23 (residues 315–363) as
the second target. Previous studies have shown that UBA2
Figure 1. Photoaffinity probes with crosslinking groups attached to
Ala46. A) Phenyl-azide-based ubiquitin probes 1, 2, and 3. B) The
diazirine-based ubiquitin probes 1’, 2’, 3’, 2’’, and 3’’.
preferentially binds Lys48-linked diUb (K = 11.5 mM) over
d
Lys63-linked diUb (K = 143.6 mM) or monoUb (K =
d
d
[
23]
5
40 mM).
In our study, UBA2 was prepared through
chemical synthesis. After folding, synthetic UBA2
(2.0 equiv, 5.0 mm) was reacted with 1, 2, or 3 (1.0 equiv,
2.5 mm) under the same photo-crosslinking conditions as
described above for tUIM. The corresponding reaction
mixtures were resolved with SDS-PAGE, and we found that
2 and 3 crosslinked to UBA2 with almost the same efficiency
(Figure 3B). Therefore, we concluded that aryl azide is not
a suitable crosslinking group for developing photoaffinity
probes to selectively capture Ub-binding proteins.
with a 49% yield of isolated product (Figure 2B). The
product (7) was treated with TFA to remove Aux, and this
was followed by AgOAc treatment to remove Acm, which led
[17]
to intermediate 8 with a yield of 79%.
Subsequently,
ligation between 8 (1.0 equiv) and 4 (2.1 equiv) was per-
formed to generate 9. Selective modification of the Cys
residues of 9 was conducted with 1-(4-azidophenyl)-2-bro-
moethan-1-one to produce probe 3 with an overall yield of
[18]
1
4%.
The identity and purity of 3 were characterized
To avoid non-selective crosslinking, the alternative photo-
[22]
through analytical RP-HPLC and electrospray-ionization
mass spectrometry (ESI-MS; Figure 2C,E and the Supporting
Information). Circular dichroism (CD) spectroscopy was used
to characterize the secondary structure of 3 (Figure 2G), and
the probes showed absorption at both 208 and 226 nm. These
spectra showed very similar patterns to that of monoubiquitin
affinity group diazirine was considered.
The diazirine-
based photoaffinity probes 1’, 2’, and 3’ (Figure 3B) were
obtained in a similar manner to 1, 2, and 3 through protein
chemical synthesis (Figure 2A,C,F and the Supporting Infor-
mation). Probes 1’, 2’, and 3’ were used for the crosslinking
reaction. Using SDS-PAGE to analyze the photo-crosslinking
reactions with Rap80 tUIM, we observed that 1’, which
contains only one Ub unit, did not crosslink with tUIM
(Figure 4A left). For probes containing two Ub units, only 3’
specifically crosslinked with tUIM (Figure 4A, left). Impor-
tantly, when we added increasing doses of Lys63-linked diUb
into the photo-crosslinking mixtures of 3’ with the tUIM, we
observed a dose-dependent decrease in crosslinking product
(Figure 4C). This result indicates that Lys63-diUb competes
(
monoUb), thus indicating that the Ub units in 3 retain the
[
19]
correct globular fold.
We next examined the ability of 1, 2, and 3 to capture
model Ub-binding proteins. The tandem ubiquitin-interacting
motif (tUIM) in human protein Rap80 (residues 80–121) was
chosen as the first target. Cohen et al. have shown that tUIM
preferentially binds to Lys63-linked diUb (binding constant
K = 17.6 mM) over Lys48-linked diUb (K = 157 mM) or
d
d
[
20]
monoUb (K = 520 mM).
We therefore expected 3 to
with tUIM labeling by 3’ (2.5 mM) with IC = 57.5 mM, a value
d
50
[20]
selectively capture more tUIM than 1 or 2. To test this
hypothesis, we prepared tUIM through chemical synthesis.
After folding the synthetic tUIM, we mixed tUIM (2.0 equiv,
that matches the reported 17.6 mM binding constant (K ).
d
In addition, we tested the photo-crosslinking efficiency of 3’
(2.5 mm) with tUIM and found that tUIM crosslinks with 3’ in
a dose-dependent manner without observing any cross-link-
5
.0 mm) with 1, 2, or 3 (1.0 equiv, 2.5 mm). The mixtures were
2
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Figure 2. Chemical synthesis of probes 3 and 3’. A) The synthesis route. Acm=acetamidomethyl. B) Auxiliary-assisted ligation monitored by
analytical RP-HPLC. 5’ is the 4-mercaptophenylacetic acid (MPAA) thioester Ub [Met1-Phe45]-MPAA. C–D) Analytical RP-HPLC chromatograms of
purified 3 and 3’. E–F) ESI-MS of 3 and 3’. G) CD spectra of 3 and 3’ compared with monoUb.
Figure 3. A,B) SDS-PAGE analysis of the photo-crosslinking reactions
with Rap80 tUIM or hHR23a UBA2 using probes 1, 2, and 3. *
indicates monomeric streptavidin. C) LC-MS/MS analysis of the cross-
Figure 4. A) Photo-crosslinking reactions of 1’, 2’ and 3’ with Rap80
linked peptide fragments of 1, 2, and 3 with tUIM. D) Proposed
tUIM or hHR23a UBA2. B) Photo-crosslinking of the probes with UBA2
mechanism of unwanted phenyl azide crosslinking. Proteins were
was selectively inhibited by Lys48-diUb but was unaffected by mono-
separated with 15% SDS-PAGE and analyzed through Coomassie blue
Ub. Proteins were separated by 15% SDS-PAGE and analyzed through
staining.
Coomassie staining. * indicates monomeric streptavidin. C) Lys63-
diUb inhibited photo-crosslinking of 3’ with Rap80 tUIM. D) Concen-
tration-dependent labeling of tUIM by 3’.
ing of 3’ with two tUIM molecules. The labeling saturated at
approximately 10 mM tUIM (EC = 2.8 mM) (Figure 4D),
5
0
thus indicating that the introduction of photoaffinity groups
did not substantially affect Lys63-diUb binding to tUIM.
Additionally, no crosslinking was detected between 3’ and
Lys63-linked diUb (50- to 100-fold more than tUIM) in this
experiment. These observations strongly suggest that the
crosslinking of 3’ exhibits high protein selectivity. Probes 2’’
and 3’’ (Figure 1B, Figure S31 in the Supporting Information),
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which have only one photoaffinity label, were also chemically
synthesized for the photo-crosslinking reactions, and the
photo-crosslinking capture efficiency and specificity of this
probe set were similar to those for the probes with two
photoaffinity labels (Figure S31). Because the synthesis yields
of 2’’ and 3’’ were relatively low (7% for 2’’ and 5% for 3’’,
Figure S17), 2’ and 3’ were chosen for the following experi-
ments.
To further confirm the crosslinking selectivity of the
diazirine-based photoaffinity probes, UBA2 was tested. We
found that UBA2 was only captured by probe 2’, and no cross-
linking was observed for 1’ or 3’ (Figure 4A, right). The
labeling of UBA2 is subject to competition by native Lys48-
diUb. Importantly, this competition was specific, because the
presence of monoUb did not interfere with labeling (Fig-
ure 4B), thus verifying that the diUb probes exhibit linkage-
dependent selectivity for Ub-binding proteins.
the deubiquitinase UBP1, which is known to modulate Lys48
[
25]
polyUb-modified P53 (Figure 5C).
The above results
indicate that the diazirine photoaffinity probes selectively
crosslink with Ub-binding proteins.
In summary, photoaffinity probes with oligoubiquitin
skeletons were developed as new tools for the proteomic
identification of Ub-binding proteins. We have demonstrated
that these probes can be efficiently prepared through total
chemical synthesis and can be precisely functionalized with
isopeptide linkages and photo-crosslinking groups. An impor-
tant finding of our work is that probes with different diUbs
exhibit altered selectivity for capturing proteins, thus indicat-
ing the potential for linkage-dependent Ub probes that could
be applied to identifying new Ub-binding proteins for
subsequent biochemical and functional studies.
Finally, we examined whether diazirine-based probes Acknowledgements
could be used to profile Ub-binding proteins in real pro-
teomes. Probes 2’ and 3’ (2.5 mm) were added to 293F cell
lysates (2 mgmL ). The samples were irradiated with 365 nm
This study was supported by NSFC grants (Nos. 81172893 and
À1
91442121 for C.-L. Tian, No. 21402206 for J.-S. Zheng, Nos.
21532004, 81621002, and 21621003 for L. Liu) and Grants
from the Ministry of Science and Technology (2015CB910103
and 2013CB932800).
UV light for 10 min, and streptavidin-coupled beads were
used to enrich for the putative targets. After resolution by
SDS-PAGE (Figure 5A) and in-gel digestion with trypsin, the
proteins enriched by streptavidin were characterized by LC–
MS/MS. After subtracting the control hits (irradiated cell
lysates without any probe) from the samples, we identified Conflict of interest
24 proteins bound to 2’ and 423 proteins bound to 3’
Figure 5B and the Supporting Information). Analysis
3
(
The authors declare no conflict of interest.
revealed that 280 proteins were captured by both 2’ and 3’,
including many known Ub-binding proteins, as well as Ub-
related enzymes such as RABGEF1, Prp8, HDAC6, USP5,
Keywords: native chemical ligation · photoaffinity labelling ·
protein modifications · protein synthesis · ubiquitination
[4]
and USP13. Furthermore, 143 proteins were captured by
only 3’ but not 2’ (Figure 5C), thus suggesting a binding
preference for Lys63-linked polyUbs, as has been validated
by studies of Rap80, Optineurin, and Tax1BP1, which are
[
20,24]
known Lys63-polyUb binders.
Moreover, we identified
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Manuscript received: November 30, 2016
Final Article published: && &&, &&&&
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Communications
Chemie
Communications
Photoaffinity Labelling
Spoilt for choice: Diazirine-based photo-
affinity probes for ubiquitin (Ub)-binding
proteins based on K48-and K63-linked
diubiquitin were developed. Diazirines
were shown to be preferable to aryl azides
as the photo-crosslinking group, since
they decrease non-selective capture. Dif-
ferent capture selectivity was observed for
the probes with different types of linkage,
thus indicating the potential to develop
linkage-dependent probes for selectively
profiling Ub-binding proteins.
J. Liang, L. Zhang, X.-L. Tan, Y.-K. Qi,
S. Feng, H. Deng, Y. Yan, J.-S. Zheng,
L. Liu, C.-L. Tian*
&&& — &&&
Chemical Synthesis of Diubiquitin-Based
Photoaffinity Probes for Selectively
Profiling Ubiquitin-Binding Proteins
6
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ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
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