Genetically Encoded 2-Aryl-5-carboxytetrazoles for Site-Selective Protein Photo-Cross-Linking

Article abstract of DOI:10.1021/jacs.7b02615

The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein-protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein-protein interactions and their interfaces in living cells.

Full text of DOI:10.1021/jacs.7b02615

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Communication
Genetically encoded 2-aryl-5-carboxytetrazoles
for site-selective protein photo-cross-linking
Yulin Tian, Marco Paolo Jacinto, Yu Zeng, Zhipeng Yu, Jun Qu, Wenshe R. Liu, and Qing Lin
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 19 Apr 2017
Downloaded from http://pubs.acs.org on April 19, 2017
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Genetically encoded 2-aryl-5-carboxytetrazoles for site-
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†
†
fl
†,#
^
fl
†,*
Yulin Tian, Marco Paolo Jacinto, Yu Zeng, Zhipeng Yu, Jun Qu, Wenshe R. Liu, Qing Lin
†Department of Chemistry, State University of New York at Buffalo, Buffalo, New York 14260, United States
flDepartment of Chemistry, Texas A&M University, College Station, Texas 77845, United States
^Department of Pharmaceutical Sciences, State University of New York at Buffalo, Buffalo, NY 14260, United States
Supporting Information Placeholder
Chart 1. Genetically encoded photo-cross-linkers.
ABSTRACT: The genetically encoded photo-cross-linkers
promise to offer a temporally controlled tool to map transient and
dynamic protein-protein interaction complexes in living cells. Here
we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-
lysine analogs (ACTKs) and their site-specific incorporation into
proteins via amber codon suppression in E. coli and mammalian
cells. Among five ACTKs investigated, N-methylpyrroletetrazole-
lysine (mPyTK) was found to give robust and site-selective photo-
cross-linking reactivity in E. coli when placed at an appropriate site
at the protein interaction interface. A comparison study indicated
that mPyTK exhibits higher photo-cross-linking efficiency than a
diazirine-based photo-cross-linker, AbK, when placed at the same
It is noted that a biocompatible proximity-driven nucleophilic
location of the interaction interface in vitro. When mPyTK was
0
substitution reaction between
fluoroacetyllysine and cysteine was reported recently for mapping
a genetically encoded N -
introduced into the adapter protein Grb2, it enabled the photo-
capture of EGFR in a stimulus-dependent manner. The design of
mPyTK along with the identification of its cognate aminoacyl-
tRNA synthetase makes it possible to map transient protein-protein
interactions and their interfaces in living cells.
1
1
the protein-protein interaction interface. However, the occurrence
of native cysteine at the protein-protein interaction interface is
1
2
rather rare. Herein, we report the synthesis of a panel of ACT-
lysine analogs (ACTK, Chart 1), and the identification of a new
ACTK-specific pyrrolysyl-tRNA synthetase for site-specific
incorporation of ACTK into proteins in E. coli and in mammalian
cells. One of the ACTK analogs, mPyTK, exhibited robust and site-
selective photo-cross-linking of a GST dimer in bacteria. In a
comparison study, mPyTK showed significantly higher cross-
linking efficiency than AbK when both are incorporated at the same
location of GST. Moreover, the mPyTK-encoded adapter protein,
Grb2, showed a stimulus and position-dependent capture of its
transient interaction partner, epidermal growth factor receptor
(EGFR), in mammalian cells.
To map dynamic protein-protein interactions in living cells, a
powerful chemical strategy involves the use of genetically encoded
photo-cross-linkers that permanently link transient protein-protein
interaction complexes with a burst of light. Based on the structures,
the reported genetically encoded photo-cross-linkers contain one of
1
the three moieties: phenyl azide such as pAzF, benzophenone such
2
3,4
as pBpa, and diazirine such as AbK and their derivatives (Chart
1
5
-8
). While these genetically encoded photo-cross-linkers have
Since pyrrolysyl-tRNA synthetase (PylRS) and its variants have
shown tremendous versatility in charging various lysine derivatives
into proteins site-selectively in bacteria, yeast and mammalian
proven to be valuable in the study of protein structure and function,
they invariably cross-link with their interacting protein partners
with no selectivity for any particular residue as the photo-generated
reactive intermediate, i.e., the nitrene from phenyl azide, the
diradical from benzophenone and the carbene from diazirine,
inserts into a proximal C-H bond with appropriate distance and
13
cells, we decided to append ACT motif onto the c-amino group
via simple acylation reaction. For the synthesis of ACTK analogs
1
-4 in Chart 1, the key intermediate, ethyl 2-aryl-2H-tetrazole-5-
II
carboxylate, was obtained through Cu -catalyzed cross-coupling of
9
angle, making it difficult to predict a prior suitable positions for
ethyl 2H-tetrazole-5-carboxylate with the phenylaryliodonium
installation of the photo-cross-linker. In addition, the tandem mass
spectrometry-based mapping of the interaction interface is
complicated as any residue from the interacting protein partner can
potentially participate in the photo-cross-linking.
14
salt (Schemes S1-S4 in Supporting Information). For PhTK (5),
the Kakehi tetrazole synthesis was followed to give the ethyl 2-
phenyl-2H-tetrazole-5-carboxylate intermediate (Scheme S5 in
15
Supporting Information). Subsequent hydrolysis and coupling with
Fmoc-lysine•HCl followed by removal of the protecting group
afforded the ACT-lysine analogs 1-5 (Chart 1). To identify PylRS
mutants that efficiently charge mPyTK (1), an MmPylRS library, in
which four residues surrounding the N-methylpyrrole-lysine side
chain based on the crystal structure of M. mazei PylRS in complex
Recently, we reported a new photo-affinity label based on 2-aryl-
5-carboxytetrazole (ACT) with a size similar to benzophenone,
which cross-links its target proteins via addition with a proximal
1
0
nucleophile near the active site. Since ACT exhibits ligand-
dependent selective photo-cross-linking, we envisioned that ACT
may also serve as a new class of genetically encoded photo-cross-
linkers for mapping transient protein-protein interaction interfaces.
16
with Pyl-AMP (Y306, L309, C348 and Y384; Figure 1a) were
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(
Figure 2b), indicating that the ACT moiety is responsible for dimer
Since ACT photo-reacts with proximal nucleophilic residues on
proteins, we sought to determine which nucleophilic residues on
the opposite GST monomer might react with the photo-generated
carboxy-nitrile imine intermediate. To this end, we built a model of
the GST-E52mPyTK and surveyed the chemical environment
surrounding mPyTK. Four nucleophilic residues (E92, M133, C139
and K141) were identified that are located 2.8œ13.0 Å from the
electrophilic nitrile imine carbon (Figure 3a). To determine which
one of these four residues participates the cross-linking reaction,
we mutated these residues to alanine and examined the photo-cross-
linking activity of the resulting mutants. We found the Glu92‰Ala
mutation completely abolished the covalent dimer formation while
other mutations had no effect (Figure 3b). This result is consistent
with the proximity-driven reactivity as E92 is closest to mPyTK
with a calculated distance between the carboxylate oxygen and the
nitrile imine carbon of 2.8 Å (Figure 3a). Similar results were
obtained when the alanine scan was conducted with the GST-E52-
FTK mutant (Figure S7 in SI). We propose a photo-cross-linking
mechanism in which the E92 carboxylate undergoes nucleophilic
addition to the photo-generated carboxy-nitrile imine followed by
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cross-linking. For comparison, we expressed the GST-E52AbK
-
1
mutant using the wild-type PylRS with a yield of 2.5 mg L and
examined its photo-cross-linking reactivity. To our surprise, AbK
exhibited very weak reactivity as the dimer band was detected only
by western blot (Figure S5 in SI) but not Coomassie blue (Figure
2
when E. coli cells expressing GST-E52mPyTK were directly
photoirradiated, which could be attributed to higher intracellular
concentration of the GST mutant (Figure 2c and Figure S6 in SI).
Since mPyTKRS can charge other ACTKs into proteins site-
selectively, we expressed SjGST mutants carrying PyTK, FTK,
TTK and PhTK, respectively, at position-52 and compared their
photo-cross-linking efficiency in E. coli cells. Based on western
blot analysis, only mPyTK, FTK and TTK showed the cross-linked
dimer with the efficiency order of mPyTK > TTK > FTK (Figure
2d), presumably due to the highest electron density at N-methyl-
pyrrole ring, which helps to stabilize the photo-generated carboxy-
4
b). Interestingly, a higher cross-linking yield (~79%) was obtained
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nitrile imine and increase its life-time in biological media.
a)
1
,4-acyl shift (Figure 3c). The rearranged cross-linked structure
was supported by tandem mass spectrometry data in which the two
fragment ions derived from the two discrete cleavage pathways
were positively identified (Figure S8 in SI). For comparison, we
performed photo-cross-linking studies with the same set of alanine
mutants of the GST-E52AbK (E92A, M133A, C139A and K141A).
We did not observe prominent attenuation in GST dimer formation;
unexpectedly, two alanine mutants (M133A and K141A) showed
greater dimer formation than the GST-E52AbK alone (Figure S7 in
SI), presumably due to a remodeling of the interaction interface that
9
alters the distance and/or angle of a suitable proximal C-H bond.
b)
To examine whether the genetically encoded mPyTK can capture
proteinœprotein interaction complexes in mammalian cells, we first
confirmed that when mPyTKRS was expressed in HEK293T cells,
it allows site-selective incorporation of mPyTK into mCherry-
TAG-EGFP based on confocal fluorescence microscopic analysis
E52mPyTK
E52mPyTK E92A M133A C139A
K141A
UV
MW
-
+
-
+ -
+
-
+
-
+
(kDa)
(Figure S9 in SI). We then introduced mPyTK into Grb2, an adaptor
(
GST)2
55
protein that links phosphorylated EGFR to the Ras signaling
*
1
8
pathway through guanine nucleotide exchange factor Sos and was
used previously for evaluating the genetically encoded photo-cross-
GST
19
2
5
linkers in mammalian cells. Inspection of crystal structure of the
Grb2 SH2 domain in complex with a phosphotyrosine-containing
2
0
c)
heptapeptide ligand revealed that 9 residues surrounding the
ligand, A91, D104, V105, Q106, F108, K109, L111, W121 and
N143, could be mutated to mPyTK for potential photo-cross-
linking with the SH2 ligands such as the cytoplasmic domain of
EGFR (Figure 4a). Thus, HEK293T cells were co-transfected with
=
Pyl
pCMV-mPyTKRS-tRNA CUA encoding mPyTK-specific PylRS
and tRNA CUA, pCMV6-Grb2-myc-DDK encoding either wild-
Pyl
(GST)2
type or amber mutant with TAG codon substituted at any of the 9
positions with a C-terminal myc-DDK tag, and pcDNA3-EGFR-
EGFP encoding full-length EGFR and a C-terminal EGFP tag, and
protein expressions were carried out in DMEM medium
supplemented with 10% FBS and 1 mM mPyTK. The cells were
starved for 12 h before EGF stimulation and subsequent
photoirradiation on ice. The cells were then lysed and the lysates
were treated with protein tyrosine phosphatase 1B to hydrolyze the
phosphotyrosine to obviate non-covalent Grb2-interacting protein
complexes. The cross-linked Grb2-interacting proteins were
immunoprecipitated with anti-Flag antibody and analyzed by
sequential western blots using anti-myc and anti-EGFR antibodies.
The cross-linked Grb2xEGFR complex was detected for 8 out 9
Grb2-mPyTK mutants; the D104mPyTK mutant gave the highest
photo-cross-linking yield followed by the V105mPyTK,
Q106mPyTK and N143mPyTK mutants (Figure 4b and Figure S10
in SI). The clustering of the four mutants (D104, V105, Q106 and
N143) in the same region of Grb2 SH2 domain suggests that the
Figure 3. Identifying the mPyTK photo-cross-linking site in GST. (a)
A close-up view of the nucleophilic residues from the opposite GST
monomer (colored in gray) surrounding mPyTK in GST monomer
(
colored in yellow). The side chains of proximal resides (E92, M133,
C139 and K141) are rendered in tube model. (b) Coomassie blue
stained SDS-PAGE gel showing UV-dependent cross-linking of the
GST-mPyTK alanine mutants. Asterisk indicates an impurity derived
from Ni-NTA affinity purification. The proteins were photoirradiated
with a handheld 302-nm UV lamp on ice for 15 min before SDS-
PAGE. (c) Proposed mechanism for mPyTK-mediated photo-cross-
linking of GST dimer. The two cleavage pathways are marked with
blue and red dash lines on the cross-linked structure (see Figure S8 in
SI for details).
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mPyTK photo-cross-linker in these mutants may react with the
ASSOCIATED CONTENT
Supporting Information
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same nucleophilic residue on EGFR across the interaction
interface. Moreover, the photo-cross-linking of EGFR is EGF
stimulation and photoirradiation-dependent and is mediated by
mPyTK as the wild-type Grb2 did not exhibit covalent capture of
EGFR (Figure S11A in SI). The highest photo-cross-linking yield
for Grb2-D104mPyTK was observed when cells were stimulated
with EGF for 15 min (Figure S11B in SI), indicating that the
Grb2xEGFR interaction is transient and dynamic, resembling
Supplemental figures and table, synthetic schemes, experimental
procedures, characterization of new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
2
1
Corresponding Author
some other known EGF-dependent protein-protein interactions.
a)
Qing Lin (qinglin@buffalo.edu).
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Present Address
#Sichuan University College of Chemistry, China.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We gratefully acknowledge the NIH (GM085092 to Q.L., CA
1
61158 to W.R.L.) for financial support. We thank Prof. John
Koland at University of Iowa for providing the pcDNA3-EGFR-
EGFP plasmid, and Carlo Ramil in Q.L. lab for cloning assistance.
b)
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Figure 4. Photo-cross-linking of EGFR by the mPyTK-encoded Grb2
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In summary, we have synthesized a panel of 2-aryl-5-carboxy-
tetrazole-based photo-cross-linkers and evolved a polyspecific
pyrrolysyl-tRNA synthetase variant that charges these ACT-lysine
analogs site-selectively into proteins in E. coli. One of the most
reactive, genetically encoded photo-cross-linkers, mPyTK, allowed
site-selective photo-cross-linking of a GST dimer in vitro and in E.
coli cells. In comparison studies, mPyTK exhibited a significantly
higher efficiency than AbK when placed at the same location of the
GST dimer interface. Moreover, mPyTK enabled covalent capture
of the transient Grb2-interacting protein partner in mammalian cells
in a stimulus-dependent manner. In view of their higher photo-
cross-linking yields and unique cross-linking mechanism, these
genetically encoded ACT-lysines should offer a powerful chemical
tool to map transient protein-protein interactions underlining signal
transduction pathways. Because tandem MS analysis revealed the
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