Photoaffinity palladium reagents for capture of protein-protein interactions

Article abstract of DOI:10.1039/c9ob01048c

Protein-protein interactions (PPIs) are indispensable in almost all cellular processes. Probing of complex PPIs provides new insights into the biological system of interest and paves the way for the development of therapeutics. Herein, we report a strategy for the capture of protein-protein interactions using photoaffinity palladium reagents. First, the palladium-mediated reagent site specifically transferred a photoaffinity modified aryl group to the designated cysteine residue. Next, the photoaffinity group was activated by UV radiation to trap the proximal protein residue for the formation of a crosslink. This strategy was used to capture the PYL-ABA-PP2C interaction, which is at the core of the abscisic acid (ABA) signalling pathway. Our results indicated that this palladium-mediated strategy can serve as an alternative for incorporating an increasing number of diverse substrates for protein crosslinking through cysteine modifications and can be explored for use in mapping protein-peptide or protein-protein interaction surfaces and in trapping potential interacting partners.

Full text of DOI:10.1039/c9ob01048c

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Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
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Photoaffinity Palladium Reagents for Protein-Protein Interaction
Capture
Qizhen Zheng,a Zhengyuan Pang,a Jingwei Liu,a Yi Zhou,a Yang Sun,a Zheng Yin *a and Zhiyong Lou *b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Protein-protein interactions (PPIs) are indispensable in almost all unnatural amino acids (UAAs) photocrosslinker, which can be
cellular processes. Probing complex PPIs provides new insights into genetically incorporated into proteins by mutant
a
the biological system of interest and paves the way for the aaRS/tRNACUA pair.9 The other is the bi-functional
development of therapeutics. Herein, we report a strategy for photocrosslinkers that is incorporated through natural amino
protein-protein interactions capture using photoaffinity palladium acid modifications.10-12 Compared to the genetic code
reagents. First, the palladium-mediated reagent site specifically expansion strategy use for incorporating photocrosslinking
transferred a photoaffinity modified aryl group to the designated UAAs, the natural amino acid modification method offers a
cysteine residue. Next, the photoaffinity group was activated by UV more direct way to incorporate a series of photoaffinity groups.
radiation to trap the proximal protein residue for the formation of
Among all natural amino acids, cysteine is truly unique due to
crosslink. This strategy was used to capture the PYL-ABA-PP2C its diverse chemical reactivity and relatively low abundance in
interaction, which is at the core of the abscisic acid (ABA) signalling proteins.13-15 However, the currently reported cysteine
pathway. Our results indicated that this palladium-mediated modification-based bi-functional photocrosslinkers are mainly
strategy can serve as an alternative for incorporating an increasing maleimide derivatives with questionable product stability.10, 16
number of diverse substrates for protein crosslinking through By contrast, palladium oxidative addition complexes can be
cysteine modifications and can be explored for use in mapping used to modify cysteine, creating promising photocrosslinkers
protein-peptide or protein-protein interaction surface and in due to their broad substrate scope and benign reaction
trapping potential interacting partners.
conditions.16, 17 This rapid, chemo- and regioselective method
can readily convert cysteine to sulfhydryl-aryl bioconjugates,
which have been proved to be stable towards oxidants, bases,
acids and external thiol nucleophiles.16 Therefore, we
anticipated the ability to exploit this facile cysteine modification
reaction to introduce various photoaffinity groups with linkers
of different lengths at target positions on the protein of interest.
With the photoaffinity group modified protein, we expected to
capture interacting proteins upon UV exposure.
Introduction
Protein-protein interactions serve fundamental role in many
cellular processes.1 The discovery and elucidation of PPIs
functions are essential for comprehending signalling pathways
and guiding research on drug discovery.2, 3 To date, numerous
techniques, both in vitro and in vivo, have been developed and
have greatly expanded the toolkit used to understand complex
PPI networks.4-6 Among these methods, chemical crosslinking,
especially photocrosslinking, has been increasingly favoured in
Results and discussion
recent years due to its unparalleled ability to trap weak and Design and synthesis of photoaffinity palladium reagents
transient interactions.7, 8 A stable covalent crosslinking product
To employ a palladium-mediated cysteine conjugation reaction
enables efficient purifications and MS identifications. Chemists
for protein-protein interaction capture, a series of photoaffinity
have developed various photocrosslinkers for protein
compounds containing a carbon-halogen bond was designed
crosslinking, and two types of them are widely used. One is the
and synthesized. There are three commonly used photoaffinity
groups: azides, diazirines and diaryl ketones (benzophenone
derivatives), which can generate reactive species of nitrene,
a. Center of Basic Molecular Science, Department of Chemistry, Tsinghua University,
19
carbene and bi-radical molecule, respectively.18,
Azides,
Beijing 100084, China. Email: yinzheng@mail.tsinghua.edu.cn
b. Collaborative Innovation Center of Biotherapy and MOE Key Laboratory of Protein
Science, School of Medicine, Tsinghua University, Beijing 100084, China.
Email: louzy@mail.tsinghua.edu.cn
especially aryl azides, have
a maximum absorption at
approximately 260 nm, which can cause substantial damage to
biomacromolecules, restricting their use in photoaffinity
labelling.18 In contrast, diazirines and diaryl ketones are more
†Electronic
Supplementary
Information
(ESI)
available:
See
DOI: 10.1039/x0xx00000x
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widely employed for photoaffinity labelling due to their good
Journal Name
Then we tested the activity of A, C and D. Similar to B, all the
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DOI: 10.1039/C9OB01048C
stability, suitable absorption wavelength (approximately 360 target conjugates were readily obtained (Fig. 2), indicating
nm) and high reactivity in hydrophobic environments.18 broad substrate scope of the reaction. Meanwhile, it is notable
Therefore, we chose these two photoaffinity groups for further to mention that all these palladium reagents were very stable.
Ⅱ
study. Four palladium reagents (Fig. 1), Pd -I-aryl- Powders stored at -80°C for more than one year, and stock
Ⅱ
trifluoromethyldiazirine (A), Pd -I-aliphatic diazirine (B, C) and solutions stored at -80°C for more than two months could still
Ⅱ
Pd -I-benzophenone (D), using 2-dicyclohexlphosphino-2’,6’- maintain their reactivity (Fig. S2, ESI†).
diisopaopoxybiphenyl (RuPhos) as ligand were prepared from
their corresponding aryl iodide precursors, 1A, 1B, 1C and 1D,
respectively, via a one-step reaction. These oxidative addition
products were stored at -80°C.
Fig. 1 Structures of photoaffinity palladium reagents containing
different photoactivatable groups synthesized and used in this study.
Peptide model reactions
The high activity and selectivity of palladium reagents for
cysteine modification have been previously validated by
research.16 An unfolded model peptide that contains all reactive
groups such as amino, carboxyl, hydroxyl and sulfhydryl, was
used to test the reagent activity and optimize the reaction
conditions. Solid palladium reagents were dissolved in a 50%
CH3CN and 50% DMSO co-solvent to a concentration of 2.5 mM,
which was subsequently aliquoted and stored at -80°C. B was
used as a model compound to study the reaction conditions. 10
μM peptide and 20 μM B were incubated at room temperature
for 5 min. The corresponding sulfhydryl-aryl bioconjugate was
obtained with a maximum yield that exceeded 95% within a
broad pH range (6.5-8.5) in a variety of buffers (Table 1). With
this robust conversion, we decided to use the conditions (20
mM Tris-HCl buffer, 5% organic solvent, pH 8.0) for future
experiments.
Fig. 2 HPLC-MS analysis of cysteine-containing peptide modified by
photoaffinity palladium reagents. The HPLC analysis (a) of compound B
reacting with model peptide in 20 mM Tris-HCl buffer (pH 8.0). The
corresponding ESI-MS analysis of model peptide before (b) and after (d)
modification. The full peptide mass was found at 1647.750 Da, and the
expected mass of peptide-B conjugates was found at 1821.830 Da. All
other three photoaffinity palladium reagents, compound A (c), C (e),
and D (f), could reacted with model peptide, and the found full mass of
corresponding sulfhydryl-aryl conjugates were 1831.776 Da, 1835.847
Da and 1828.806 Da, respectively.
Cysteine-containing protein modification
Table 1 Peptide model reactions
We next sought to explore protein modification.
PYR/PYL/RCARs proteins (hereafter refers to as PYLs) were
chosen as targets because of their fundamental roles in the ABA
signalling pathway.20, 21 ABA, a plant hormone responsible for
plant growth, modulates the response to environmental
stress.22 Structure analysis revealed that PYLs possess a
hydrophobic ABA binding pocket that forms a conserved ‘helix-
grid’ fold. Two highly conserved loops located in the binding
pocket adopt a closed conformation upon ABA binding and form
a gate-latch interface for interactions with proteins such as type
2C protein phosphatases (PP2Cs).23 Among all the PYLs, we
chose PYR1, a major member of the PYL family, for our study.24,
25 It has been well established that PYR1 can robustly inhibit the
PP2C protein ABI1 and HAB1 in the presence of ABA, which
makes it ideal to verify the feasibility of our method.20, 26 Wild-
type PYR1 has three cysteine residues: C30, C65 and C77. These
cysteine residues are all located on the surface of PYR1 and are
Entry
Buffer
pH
Organic
Solvent %
Product %
1
2
3
4
5
6
7
8
9
20 mM MES
20 mM Tris
20 mM Tris
20 mM Tris
20 mM Tris
50 mM Tris
100 mM Tris
20 mM HEPES
20 mM
Na2HPO4/NaH2PO4
20 mM Tris
20 mM Tris, 50 mM
NaCl
6.5
7.0
7.5
8.0
8.5
7.5
7.5
7.5
7.5
5
5
5
5
5
5
5
5
5
96
94
96
96
96
94
92
96
96
10
11
7.5
7.5
1
5
81
64
2 | J. Name., 2012, 00, 1-3
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potentially susceptible to palladium reagents. To avoid multiple modified after 20 min of palladium reagent treatment at room
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DOI: 10.1039/C9OB01048C
modifications, we mutated all cysteines in the PYR1 into serine temperature, and no corresponding sulfhydryl-aryl conjugate
and obtained a PYR1 that contains no cysteine, referred to products could be detected for PYR1S, suggesting the chemo-
herein as PYR1S. After analysing the crystal structure of PYR1- selectivity of these reagents (Fig. S3, ESI†).
ABA-HAB1 (Fig. 3a), we chose five PYR1 positions, H60, K63,
Photocrosslinking analysis of PYR1 and ABI1 interaction
R116, T162 and L166, which are all on the interaction surface,
for testing.
Finally, the modified proteins were used to trap the interacting
The Flag-GST-tagged fusion proteins PYR1S and PYR1S- protein, ABI1, in the presence of ABA. We first tested whether
H60C/K63C/R116C/T162C/L166C were expressed in E. coli and the photoaffinity groups would interfere with ABI1 binding.
purified by Glutathione-Sepharose beads. The GST tag was However, to our surprise, only a modest alteration was
cleaved by precision protease overnight at 4°C. The ABI1 observed for the three mutated proteins (Fig. 4).
phosphatase activity assay demonstrated that the PYR1
mutants PYR1S and PYR1S-H60C/K63C/R116C/T162C/L166C
displayed varying inhibitory activities towards ABI1 (Fig. 3b).
Although mutation of R116 significantly impaired target
interactions, we were delighted to find that PYR1S-H60C
showed enhanced inhibition of ABI1. Based on these findings,
we focused on PYR1S-H60C/T162C/L166C for cysteine
modification. For the initial test, 1 μM PYR1S-H60C and 10 μM
Fig. 4 ABI1 phosphatase activity assay to evaluate inhibitory profiles of
B were incubated in 20 mM Tris-HCl buffer (pH 8.0) for 5 min at
modified PYR1S-C. The reaction without ABI1 was set as negative
control and reaction without inhibitor was set as positive control. The
error bars represent the standard deviations of three independent
room temperature. The corresponding aryl conjugate product
was analysed and quantified by mass spectrometry. Substrate
could be quantitively converted to the designated sulfhydryl-
aryl conjugate product, and no remaining substrate was
observed (Fig. 3c).
experiments.
Fig. 5 Photocrosslinking analysis of modified PYR1 mutants and ABI1
interactions. ABI1-His and photocrosslinking product were indicated by
black arrow. The full length of protein gel and other protein crosslinking
analysis are summarized in supplementary information.
Fig. 3 Cysteine mutation site selection and protein modification. (a) Two
PP2Cs, HAB1 and ABI1 have the similar structure features upon binding
to PYLs, as indicated by the co-crystal structure of PYR1-HAB1 (PDB:
3QN1) and PYL1-ABI1 (PDB: 3JRQ), with PYR1 in red, HAB1 in cyan and
ABI1 in yellow. Positions chosen for cysteine mutation were labelled in
blue. (b) ABI1 phosphatase activity assay to evaluate inhibitory profiles
of various PYR1 mutants. 6 μM PYR1 mutants, 0.3 μM ABI1 and 20 μM
ABA were mixed according to protocol (see the ESI†). The reaction
without ABI1 was set as negative control and reaction without inhibitor
was set as positive control. Three replicates were made for each protein
and error bar represents standard deviation. (c) PYR1S-H60C was
quantitatively modified with diazirine after reacting with palladium
reagent B, deconvoluted mass spectra showed the full PYR1-H60C
protein molecular weight before and after modification.
With promising assay validation completed, we next
incubated modified PYR1S-C proteins with purified His6-tagged
ABI1 in the presence of ABA for 10 min and then exposed the
mixture to UV radiation. Samples without ABA or UV exposure
were used as controls. The resulting photocrosslinking products
were separated by 12% SDS-PAGE and identified by Western
blot analysis using anti-His6 and anti-Flag antibodies. We found
that, for different proteins and photoaffinity groups, the final
capturing efficiency varied significantly. As revealed in Fig. 5, for
PYR1S-H60C, all four modified versions could capture the target
ABI1 protein. In contrast, for PYR1S-T162C and PYR1S-L166C,
not all modified products worked as expected. It is worthwhile
to note that all three proteins modified by A could capture ABI1.
This is in agreement with previous findings showing that aryl-
trifluoromethyl carbene displays superior reactivity and
specificity.18 Overall, the successful formation of the PYR1-ABI1
We further explored the reaction profiles of other palladium
reagents with different PYR1S cysteine mutants under the same
conditions, along with PYR1S, which was used as a negative
control. All cysteine-containing proteins were quantitatively
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J. Name., 2013, 00, 1-3 | 3
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Journal Name
9. C. C. Liu and P. G. Schultz, Annu Rev Biochem, 2010, 79, 413-
conjugate requires both the appropriate mutation position and
the proper photoaffinity group.
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Conclusions
In summary, we have developed a straightforward strategy
using a highly efficient and selective cysteine modification
method to reliably incorporate different photoaffinity groups
for capturing interacting proteins. Four photoaffinity palladium
reagents containing three widely used photoactivatable groups,
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aryl-trifluoromethyldiazirine,
aliphatic
diazirine
and
benzophenone, with various linker lengths were synthesized
and incorporated into target proteins, indicating the
unparalleled substrate scope compared to that based on use of
unnatural amino acids. Upon UV exposure, we successfully
verified the feasibility of our method by capturing the PYR1-
ABI1 interaction in the presence of ABA in vitro. More
importantly, this strategy can be further explored in mapping
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a
promising alternative to obtain and deduce the composition
and architecture of stable protein complexes for use in studying
the PPI networks.
Conflicts of interest
There are no conflicts to declare.
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Acknowledgments
This work is supported by National Key Research and
Development Program of China (2017YFA0505203), and
National Natural Science Foundation of China (NSFC Grant No.
20151300701).
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4 | J. Name., 2012, 00, 1-3
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A straightforward strategy using palladium-mediated reagents to reliably incorporateDdOiIf:f1e0r.1e0n3V9ti/eCw9AOrtBic0le1O04nl8inCe
photoaffinity groups into peptides/proteins for interacting partners crosslinking is described.