Photocaged Quinone Methide Crosslinkers for Light-Controlled Chemical Crosslinking of Protein–Protein and Protein–DNA Complexes

Article abstract of DOI:10.1002/anie.201910135

Small-molecule crosslinkers are invaluable for probing biomolecular interactions and for crosslinking mass spectrometry. Existing chemical crosslinkers target only a small selection of amino acids, while conventional photo-crosslinkers target almost all r

Full text of DOI:10.1002/anie.201910135

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Accepted Article
Title: Photocaged Quinone Methide Cross-linkers for Light-controlled
Chemical Cross-linking of Protein-protein and Protein-DNA
Complexes
Authors: Jun Liu, Lingchao Cai, Wei Sun, Rujin Cheng, Nanxi Wang,
Ling Jin, sharon rozovsky, Ian Seiple, and Lei Wang
This manuscript has been accepted after peer review and appears as an
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201910135
Angew. Chem. 10.1002/ange.201910135
Link to VoR: http://dx.doi.org/10.1002/anie.201910135
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Angewandte Chemie International Edition
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COMMUNICATION
Photocaged Quinone Methide Cross-linkers for Light-controlled
Chemical Cross-linking of Protein-protein and Protein-DNA Complexes
#[a]
#[a]
#[a]
#[b]
[a]
[c]
[b]
Jun Liu, Lingchao Cai, Wei Sun, Rujin Cheng, Nanxi Wang, Ling Jin, Sharon Rozovsky,
[a]
[a]
Ian B. Seiple, and Lei Wang*
Abstract: Small molecule cross-linkers are invaluable for probing
cells, tissues, and whole organisms. [3] Chemical cross-linkers
react with target residues specifically. For instance, the most
commonly used cross-linkers contain homobifunctional N-
hydroxysuccinimidyl (NHS) esters to react with Lys side chain or
N-terminal amine. Cross-linkers targeting Cys, Glu, Asp, and His
have also been developed. [4] [5] [6] [7] Expanding the repertoire of
residues targetable and enabling multi-targeting ability of
chemical cross-linkers would increase the number and types of
constraints obtainable from CXMS. Recently, we reported a plant-
and-cast strategy enabling cross-link of Lys residues with His, Ser,
Thr, Tyr, and Lys side chains through sulfur-fluoride exchange
reaction, showcasing the feasibility of targeting multiple residues
via new chemistry. [8] However, a variety of amino acid residues
remain untargetable. On the other hand, conventional
biomolecular interactions and for cross-linking mass spectrometry
(
CXMS) in addressing large protein complexes and intrinsically
disordered proteins. Existing chemical cross-linkers target only a
small selection of amino acid residues, limiting the number and type
of cross-links, while conventional photocross-linkers target virtually all
residues non-selectively, complicating data analysis. Here we report
photocaged quinone methide (PQM)-based cross-linkers that are able
to multitarget nine nucleophilic residues through specific Michael
addition. In addition to Asp, Glu, Lys, Ser, Thr, and Tyr, PQM cross-
linkers notably cross-linked Gln, Arg, and Asn hitherto untargetable
by existing chemical cross-linkers, markedly increasing the number of
residues targetable with a single cross-linker. Such multiplicity of
cross-links will increase the abundance of cross-linked peptides for
CXMS identification and afford ample constraints to facilitate
structural deciphering. PQM cross-linkers were used in vitro, in E. coli,
and in mammalian cells to cross-link dimeric proteins and
endogenous membrane receptors. The cross-linker NHQM could
directly cross-link proteins to DNA, for which few cross-linkers exist.
The photoactivatable and multitargeting reactivity of these PQM
cross-linkers will substantially enhance chemical cross-linking based
technologies for studies of protein-protein and protein-DNA networks
and for structural biology.
photocross-linkers
(such
as
diazirines,
azides,
and
benzophenones) target virtually all residues non-selectively, [9] [10]
but such nonspecific chemistry often results in highly complex
cross-linked products, dramatically complicating MS data analysis.
In addition, excessive cross-linking may artificially distort protein
[1]
[2]
tertiary structures.
For DNA-protein cross-linking,
formaldehyde remains the primary reagent despite its short cross-
link distance (~2 Å) and limited reactivity with few residues. [3]
Therefore, new small molecule cross-linkers able to multi-target
different amino acid residues, especially those inaccessible to
date, with specific chemistry would be valuable for realizing the
full potential of cross-linking based technologies.
Small molecule cross-linkers have been invaluable for
studying biomolecular interactions. An emerging technology for
protein interaction and structural biology is the cross-linking mass
spectrometry (CXMS), which analyzes proteins cross-linked by
small molecule cross-linkers with tandem mass spectrometry,
affording identities and distance constraints of cross-linked
residues. [1] [2] It has been increasingly used to probe protein
interaction networks and to derive tertiary structural information of
large protein complexes and intrinsically disordered proteins,
complementing X-ray crystallography, NMR spectroscopy, and
cryo-electron microscopy. Small molecule cross-linkers are also
used for cross-linking DNA to proteins, which is a critical step for
chromatin immunoprecipitation (ChIP), a method widely used for
mapping DNA-protein interactions across eukaryotic genomes in
We report here a new series of photocaged quinone methide
(
PQM)-based small molecule cross-linkers, which integrate the
advantages of chemical cross-linkers (i.e., specific chemical
reactivity) and conventional photocross-linkers (i.e., photo-
controllability and thus potential for spatiotemporal resolution) for
cross-linking biomolecules. These PQM cross-linkers were able
to multitarget nine different amino acid residues through Michael
addition in protein-protein cross-linking. We demonstrated their
use for cross-linking proteins in vitro, in E. coli, and in mammalian
cells, and for cross-linking proteins with DNA as well.
Quinone methides (QM) are efficient Michael acceptors for
nucleophiles and have been versatile for chemical synthesis and
[11] [12] [13] [14]
chemical biology.
We recently genetically encoded an
unnatural amino acid FnbY containing a photocaged para-QM
into proteins and showed its specific reactivity toward multiple
nucleophilic residues placed in proximity. [15] The less reactive
[a]
Dr. J. Liu, Dr. L. Cai, Dr. W. Sun, Dr. N. Wang, Prof. I. B. Seiple, and
Prof. L. Wang
quinone has also been explored for protein cross-linking [16] [17] [18]
.
University of California, San Francisco, Department of
Pharmaceutical Chemistry and the Cardiovascular Research
Institute, 555 Mission Bay Blvd. South, San Francisco, CA, 94158
E-mail: Lei.wang2@ucsf.edu
We therefore reasoned that integration of photocaged highly
reactive QM into small molecule cross-linkers would enable the
desired multi-targeting ability through specific Michael addition
chemistry, as well as photocontrolled reactivity for spatiotemporal
resolution.
[b]
R. Cheng, and Prof. S. Rozovsky.
University of Delaware, Department of Chemistry and Biochemistry,
Newark, DE, 19716
We initially designed a heterobifunctional cross-linker NHQM
containing an NHS ester and a photocaged ortho-QM (o-QM) (Fig.
[c]
L. Jin
University of Florida, Department of Microbiology and Cell Science,
Gainesville, Florida 32611
1
A). Two fluorines were installed at the methyl group ortho to the
#
These authors contributed equally.
phenolic hydroxyl, which was photocaged by an o-nitrobenzyl
Supporting information for this article is given via a link at the end of
the document.
group. When added to proteins, the NHS ester will rapidly react
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1
4-3-3 (E) and 14-3-3(QQR) mutant (F) in E. coli cell lysate. 14-3-3 was
with exposed Lys residues, planting the photocaged o-QM next to
residues on interaction partners. UV release of o-nitrobenzyl
induces elimination of a fluoride ion to generate o-QM, which will
then cross-link with nearby nucleophilic side chains (Fig. 1A). To
detected by an anti-Hisx6 antibody.
NHQM cross-linking results in a short and rigid linkage, which
requires close contact of target residues for effective cross-linking.
We thus tested whether this feature could be used to readily
determine protein dimerization in vitro and in cell lysate. Three
test this design, we incubated
regulatory protein 14-3-3 (Fig. 1B)
a
homodimeric eukaryotic
with 1 mM NHQM followed
[19]
by UV illumination (l = 365 nm). Cross-linked dimer of 14-3-3 was
detected on SDS-PAGE only with NHQM addition and UV
illumination (Fig. 1C), suggesting that cross-linking was through
photo-released QM. Cross-linking of 14-3-3 could be observed on
SDS-PAGE as short as 1 min of UV exposure, and increased with
UV exposure time (Fig. 1D, Fig S1), indicating that NHQM-
mediated cross-linking could be controlled by light with temporal
resolution.
mutations ( LAE → 12QQR ) of 14-3-3 disrupt the dimer
1
2
14
14
interface to form a monomer, leading to a distinct function from
[20] [21]
WT 14-3-3.
Previously the 14-3-3(QQR) mutant was
isolated from cells and then characterized as a monomer using
size exclusion chromatography. This in vitro process is tedious,
and the results may not represent what occur in the cellular setting.
We first treated purified 14-3-3 WT and QQR mutant with various
amounts of NHQM and illuminated with UV light (Fig. 2A, 2B).
Cross-linked dimer of WT 14-3-3 increased with higher NHQM
concentration on SDS-PAGE, while 14-3-3 QQR remained a
monomer (Fig. 2C, 2D). We then applied NHQM directly to lysates
of E. coli cells expressing either 14-3-3 WT or QQR mutant.
Western blot analysis of the cell lysates clearly showed that WT
1
2
4-3-3 was cross-linked as dimer but not the QQR mutant (Fig.
E, 2F). These results show that NHQM-mediated cross-linking
could be a facile method for determining protein interactions.
Figure 1. NHQM-mediated protein cross-linking in vitro. A) Scheme showing
NHQM structure and photo-controlled cross-linking mechanism. Upon UV
activation, a highly reactive ortho-QM was generated capable of reacting with
multiple nucleophilic residues. B) Protein 14-3-3 forms a homodimer (PDB code:
4
1
N7Y). C-D) SDS-PAGE analysis of NHQM-mediated dimeric cross-linking of
4-3-3 (C) with different UV exposure time (D).
Figure 3. NHQM and NHQM3C multi-target a total of nine nucleophilic residues
in protein cross-linking. A) CXMS analysis of WT 14-3-3 cross-linked by NHQM.
B) Representative tandem mass spectrum for cross-linked peptides showing
cross-linking of Lys with Gln. Others are shown in Figures S2, S3, and S5. C)
Structure of NHQM3C and its cross-linking mechanism.
To identify which residues NHQM could cross-link, we
analyzed the cross-linked 14-3-3 protein with tandem mass
spectrometry. Nine pairs of cross-linked peptides were identified,
showing that NHQM cross-linked Lys of one peptide with Gln, Lys,
Glu, Ser, Arg, Asn, and Asp of the other peptide (Fig. 3A, 3B, S2).
These residues have nucleophilic side chains, consistent with the
reaction mechanism of o-QM. The maximum Ca-Ca distance of
NHQM cross-linker is ~22 Å considering amino acid side chain
length plus the covalently connected cross-linking agent. The Ca-
Ca distances for the majority of cross-linked residues fall within
this range, with only one slightly exceeding the limit and measured
Figure 2. Facile differentiation of dimer and monomer via NHQM cross-linking
in vitro and in cell lysate. A-B) NHQM should cross-link the WT 14-3-3 dimer
into a covalent dimeric form but not the monomeric QQR mutant, which can be
readily distinguished on SDS-PAGE or Western blot. C-D) SDS-PAGE analysis
of NHQM-mediated in vitro cross-linking WT 14-3-3 (C) and 14-3-3(QQR)
mutant (D). E-F) Western blot analysis of NHQM-mediated cross-linking of WT
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at 26.6 Å (Fig. 3A, S3). To increase cross-linker’s flexibility, we
also synthesized NHQM3C, adding three methylenes to the
spacer of NHQM (Fig. 3C). When applied to the purified WT 14-
explored whether NHQM could probe endogenous mammalian
proteins at physiologically relevant conditions. The human
mammary epithelial MCF10A cells were cultured and treated with
or without NHQM followed with light activation. EGFR
dimerization on MCF10A cells was detected only in the presence
3
-3, NHQM3C also cross-linked 14-3-3 into covalent dimers upon
UV activation (Fig. S4). Tandem mass spectrometric analysis of
the cross-linked 14-3-3 identified six pairs of cross-linked peptides, of UV and NHQM (Fig. 4).
with Lys cross-linked with nucleophilic residues including Gln, Glu,
Thr, and Tyr (Fig. S5). The cross-linking pattern of each anchor
Lys was different for NHQM and NHQM3C, and the Ca-Ca
distances for NHQM3C were longer than those for NHQM,
reflecting their differences in rigidity and length. Taken together,
these results show that NHQM and NHQM3C expanded the
repertoire of natural residues targetable with small molecule
cross-linkers to a total of nine nucleophilic residues.
We next explored potential applications of NHQM in various
biological settings, starting with cross-link of interacting proteins
in E. coli cell lysate. Thioredoxin (Trx) is
a ubiquitous
oxidoreductase regulating cellular redox through interacting with
[22] [23]
various proteins.
We expressed His-tagged Trx in E. coli,
and then applied NHQM to the cell lysate followed by UV
activation. Western blot analysis of the treated cell lysates
showed that many endogenous proteins were cross-linked to Trx
(
(
Fig. S6). Together with cross-linking of 14-3-3 in E. coli cell lysate
Fig. 2E), these results show that NHQM is compatible for cross-
linking in E. coli cell lysate. We also attempted applying NHQM
directly to E. coli cells to cross-link intracellularly expressed 14-3-
Figure 5. HoQM-mediated protein cross-linking in E. coli and mammalian cells.
A) Scheme showing HoQM structure and cross-linking mechanism. B-C)
Western blot analysis of HoQM-mediated 14-3-3 cross-linking in E. coli cells (B)
and in mammalian cells (C).
3
, but did not detect any 14-3-3 dimeric form on Western blot,
possibly because NHQM could not enter E. coli cells efficiently.
To enable efficient cross-linking inside cells, we designed a
homobifunctional cross-linker HoQM, with photocaged o-QM at
both ends (Fig. 5A). We reasoned that replacing NHS ester with
the photocaged QM would remove chemical reactivity of the
cross-linker prior to photoactivation, thus allowing it to enter cells
without causing cytotoxicity. Additionally, QM at both ends could
increase the diversity of cross-link types since QM reacts with
more residues than NHS ester. As expected, HoQM cross-linked
1
4-3-3 into dimer in vitro upon light activation with comparable
efficiency as NHQM (Fig. S10). We then incubated HoQM with E.
coli cells for 1 h, and found it cross-linked the intracellularly
expressed 14-3-3 in dimeric form upon UV illumination (365 nm)
of intact cells for 15 min (Fig. 5B), which was infeasible with
NHQM. In addition, HoQM (0.6 mM) was incubated with HEK293T
cells for 4 to 8 h without apparent toxicity; subsequent exposure
of these cells to UV light (365 nm, 10 min) resulted in dimeric
cross-linking of intracellular 14-3-3 protein (Figure 5C). These
results demonstrated HoQM’s ability to cross-link proteins inside
live bacterial and mammalian cells.
Figure 4. Photo-controlled dimeric cross-linking of EGFR on mammalian cell
surface by NHQM. A) Scheme showing the process. B) Western blot analysis
of EGFR on MCF10A cells after indicated treatment. The amine-reactive cross-
3
linker bissulfosuccinimidyl suberate (BS ) was used as a positive control.
We then tested NHQM’s cross-linking ability in mammalian
cells. We incubated NHQM with live mammalian cells expressing
the dimeric glutathione transferase (GST)[9] or 14-3-3, and
detected cross-linking of both proteins in dimeric form in response
to light, albeit in low efficiency (< 10%, Fig. S7-S8), suggesting
NHQM could enter mammalian cells but at low efficiency. We thus
tested using NHQM to cross-link proteins on mammalian cell
surface. NHQM was applied to HEK293T cells, which were
transfected with plasmids to overexpress the epidermal growth
factor receptor (EGFR). EGFR dimer was detected on Western
blot in response to UV and NHQM, demonstrating NHQM cross-
linking of the receptor with optical control (Fig. S9). We further
Reagents to cross-link proteins with DNA remain sparse,
because reactivities of most cross-linkers favor protein-protein
over protein-DNA cross-linking. Formaldehyde is the primary
reagent but fails to cross-link proteins not in close contact (2 Å)
[24] [25] [3]
with DNA.
QM can alkylate deoxynucleosides efficiently.
[26]
We thus reasoned that NHQM should be able to cross-link
protein with DNA. To test this possibility, we first incubated a
single stranded DNA binding protein (SSB) [27] with a short DNA
oligo of repeats 19(3x) or ATC(4x) which are reported to interact
with SSB. [28] [29] The SSB-DNA complex was treated with NHQM
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and UV activation, and analyzed with denaturing TBE-urea gel
shift assay. An upshifted band was only observed for the SSB-
further enhance its crosslinking efficiency. We expect that the
photoactivatable and multitargeting reactivity of these PQM cross-
linkers will be valuable for investigation of protein-protein and
protein-DNA networks and structural biology through chemical
cross-linking.
1
9(3x) or SSB-ATC(4x) complex treated with both NHQM and UV
light (Figure 6A), indicating successful protein-DNA cross-linking
mediated by NHQM. As expected, the NHS ester-based amine
3
reactive BS cross-linker did not result in SSB-DNA cross-linking.
We further investigated protein-DNA cross-linking by incubating a
natural single stranded circular viral DNA M13mp18 with the SSB
protein. [30] As shown in Figure 6B (also Fig. S11), the M13mp18
control DNA run as two isoforms in the denatured TBE-Urea gel
due to its large size, a major form remained in the well and a minor
form migrating in the gel. Only in the presence of both NHQM
treatment and UV activation was the minor form of M13mp18
upshifted into the well, suggesting it was cross-linked with SSB.
Acknowledgements
We thank Dr. Natalia Jura for MCF10A cells and Devan Diwanji
for helpful discussion. S.R. acknowledge the support of NIH
(
(
R01GM121607); L.W. acknowledges the support of the NIH
R01GM118384, RF1MH114079).
Keywords: quinone methide • photo-cross-linking• cross-linker •
DNA-protein interaction • protein-protein interaction
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1
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Entry for the Table of Contents
COMMUNICATION
Jun Liu,#[a] Lingchao Cai, Wei Sun,
Rujin Cheng,#[b] Nanxi Wang, Ling
Jin,[c] Sharon Rozovsky,[b] Ian B.
Seiple,[a] and Lei Wang*[a]
#[a]
#[a]
[a]
Page No. – Page No.
Photocaged Quinone Methide Cross-
linkers for Light-controlled Chemical
Cross-linking of Protein-protein and
Protein-DNA Complexes
Text for Table of Contents
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