A genetically encoded aza-michael acceptor for covalent cross-linking of protein-receptor complexes

Article abstract of DOI:10.1021/ja502851h

Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately tuned electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNA_CUA pairs that genetically encode three aza-Michael acceptor amino acids, N^ε- acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.

Full text of DOI:10.1021/ja502851h

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Article
A genetically encoded aza-Michael acceptor for
covalent crosslinking of protein-receptor complexes
Jennifer L. Furman, Mingchao Kang, Seihyun Choi, Yu Cao, Erik D Wold,
Sophie B Sun, Vaughn V Smider, Peter G Schultz, and Chan Hyuk Kim
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja502851h • Publication Date (Web): 20 May 2014
Downloaded from http://pubs.acs.org on May 25, 2014
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A genetically encoded aza-Michael acceptor for covalent
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Jennifer L. Furman , Mingchao Kang , Seihyun Choi , Yu Cao , Erik D. Wold , Sophie B. Sun , Vaughn V. Smider ,
1,2
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Peter G. Schultz *, and Chan Hyuk Kim *
1
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Tor-
rey Pines Road, La Jolla, California 92037, United States
2
California Institute for Biomedical Research, 11119 North Torrey Pines Road Suite 100, La Jolla, California 92037, United
States
†
These authors contributed equally to this work.
*e-mail: schultz@scripps.edu; chkim@calibr.org
ABSTRACT: Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introduc-
ing into a protein an appropriately “tuned” electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its
cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNA
pairs that genetical-
CUA
ε
ly encode three aza-Michael acceptor amino acids, N -acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and
p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-
ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a spe-
cific surface lysine ε-amino group of ErbB2, leading to near quantitative crosslinking to either purified ErbB2 in vitro or to native
cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes,
diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.
Introduction
over the position and geometry of the reactive group. Thus far,
16,17
a variety of electrophiles, including alkyl and aryl ketones,
One approach to the design of high affinity, selective small-
molecule enzyme inhibitors involves the introduction of an
electrophilic group into the inhibitor that can form a covalent
18
19
20,21
trifluoroketones, α-fluoroketones, and acrylamides
have
been introduced into proteins by this method. The use of these
UAAs to form intra- or intermolecular covalent bonds typical-
ly requires a cysteine residue in the receptor of interest due to
its relatively high nucleophilicity. However, cysteine residues
are uncommon at protein surfaces and frequently found in
1
bond with an active site nucleophile. Selectivity is achieved
through proximity of the two reactive groups in the enzyme-
inhibitor complex, or in the case of mechanism-based inhibi-
tors, by unmasking of the electrophilic moiety via enzymatic
22
2
,3
cysteine crosslinks, necessitating mutagenesis of the target
protein to introduce a unique cysteine residue at the binding
interface. Instead, an ideal group to target with these electro-
transformation. Many covalent inhibitors of hydrolases, ki-
nases, oxido-reductases, and PLP-dependent enzymes have
been developed based on fluoroketones, α-haloketones, epox-
ides, α,β-unsaturated ketones and amides, and vinyl sulfona-
23
philes is the nucleophilic ε-amino group of lysine residues,
which are relatively abundant at protein surfaces and likely to
be found at the protein-protein interface of interest. The prac-
4
-7
mides. Indeed, a significant fraction of clinically used drugs
24
8
that act on enzymes are irreversible inhibitors, with recent
examples including inhibitors of the 20S proteosome and
Bruton’s tyrosine kinase (BtK).
9
tical application of this strategy requires that the reactivity of
the electrophile be tuned so it has minimal nonspecific reac-
tions with common physiological nucleophiles (such as gluta-
thione), while maintaining the ability to react with a lysine
10
The application of similar medicinal chemistry-based strate-
gies to form irreversible protein-based agonists, antagonists, or
inhibitors is made difficult by the lack of general strategies for
25
residue in a binding partner. This can be achieved through
‘proximity effects’, in which the formation of a covalent bond
is accelerated by formation of a noncovalent complex prior to
selectively introducing electrophiles into proteins at a specific
11,12
site of interest.
Current methods include reaction of unique
26
reaction. This leads to a reduction in the entropy of activa-
cysteine or lysine residues with bifunctional N,N′-ethylene
13
14
tion which can result in large increases in reaction rates (effec-
bisacrylamide or chloroacetyl chloride electrophiles to in-
troduce acrylamides or chloroacetamides, respectively. Addi-
tionally, expressed protein ligation (EPL) has been used to
label proteins with vinyl sulfones and β-lactams to generate
5
27
tive molarities >10 M).
Herein we have investigated a series of electrophilic UAAs
which contain α,β-unsaturated amides (acrylamides) and sul-
fonamides (vinyl sulfonamides), Michael acceptors which
have been extensively used to target reactive nucleophiles in
1
5
activity-based protein profiling (ABPP) probes. Alternative-
ly, the use of evolved orthogonal aminoacyl-tRNA synthetase
(aaRS)/tRNA pairs for genetically encoding unnatural amino
acids (UAAs) provides a potentially general method for intro-
ducing electrophilic groups into proteins with precise control
4
,28-30
kinases and proteases.
These reactive groups have rela-
tively low nonspecific reactivity to other serum and cellular
proteins. Therefore, we designed and genetically encoded
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three UAAs, N -acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-
S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-
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(
phenylalanine (VSF, 3), with electrophilic groups that can
react with the ε-amino group of lysine residues in a target pro-
tein (Figure 1A). Moreover, we show that a mutant of the an-
tigen-binding fragment (Fab) of the therapeutic monoclonal
antibody Herceptin bearing 3 selectively and irreversibly la-
bels ErbB2-positive breast cancer cells under physiological
conditions.
Results and Discussion
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To test whether electrophilic UAAs can be used to selec-
tively form irreversible protein complexes, we used an αErbB2
Fab based on Herceptin and the extracellular domain (ECD) of
the oncogenic receptor tyrosine kinase ErbB2 as a model sys-
tem. Herceptin binds ErbB2 with high affinity (K ~0.1 nM),
d
leading to reduced cellular proliferation through disruption of
receptor dimerization and antibody-dependent cellular cytox-
3
1-33
icity (ADCC).
We hypothesized that incorporation of a
relatively small electrophilic UAA at a defined site in Her-
ceptin would allow covalent bond formation with a proximal
lysine ε-amino group of the ErbB2 ECD without significantly
perturbing binding affinity. Examination of the Herceptin Fab-
ErbB2 ECD co-crystal structure (PDB: 1N8Z) revealed that
there are two lysine residues (Lys569 and Lys593) at the bind-
ing interface, providing potential crosslinking sites (Figure
Figure 1. Structures of unnatural amino acids and crosslinking
sites at the Herceptin Fab-ErbB2 binding interface. (a) Structures
ε
of the electrophilic unnatural amino acids N -acryloyl-(S)-lysine
(
1), p-acrylamido-(S)-phenylalanine (2), and p-vinylsulfonamido-
(S)-phenylalanine (3). (b) The crystal structure of a Herceptin Fab
bound to the extracellular domain of ErbB2 (PDB: 1N8Z) shows
two residues in the light chain (LC) CDR3 (LC-Tyr92 and LC-
Thr93) and one residue in the heavy chain (HC) CDR3 (HC-
Gly103) of the Fab that are in close proximity to the ε-amino
groups of Lys569 and Lys593 in ErbB2 at the binding interface.
34
1
B). Two residues in the light chain (LC) complementarity
determining region 3 (CDR3) of Herceptin (LC-Tyr92 and
LC-Thr93) are within 10 Å of Lys569, and one residue in the
heavy chain (HC) CDR3 of αErbB2 (HC-Gly103) is posi-
tioned to allow potential interaction with Lys593 of ErbB2.
These residues in the Herceptin Fab gene were mutated to the
amber stop codon, TAG, generating plasmids encoding Her-
ceptin Fab-Tyr92TAG, Herceptin Fab-Thr93TAG, and Her-
ceptin Fab-Gly103TAG (Supplementary Information).
aaRS (AcrKRS, mutations: Leu270Ile, Leu274Ala,
Cys313Phe, Tyr349Phe) that incorporates 1 into proteins. Us-
ing a fluorescence-based assay in which GFP with a permis-
sive Asn149TAG mutation was co-expressed with the
Pyl
CUA
AcrKRS/tRNA
pair in the presence and absence of 1, a
2
0-fold increase in fluorescence was observed upon addition
The first UAA we explored for the covalent crosslinking of
ε
of 5 mM 1 (Supplementary Information). We note that while
finalizing these experiments, there were two concurrent and
independent reports of evolved aaRSs with distinct sequences
Herceptin Fab to ErbB2 was the alkyl acrylamide, N -acryloyl-
(S)-lysine (AcrK, 1). Synthesis of 1 was readily achieved in
one step in 54% yield (Supplementary Information). Engi-
20,21
Pyl
CUA
that incorporate 1 in prokaryotes and eukaryotes.
neered pyrrolysyl-tRNA synthetase (PylRS)/tRNA
pairs
To determine the fidelity and efficiency for incorporation of
from Methanosarcina species (barkeri and mazei) can be used
1
into proteins in E. coli, Herceptin Fab mutants with amber
to genetically encode a variety of lysine analogues, including
ε
35
ε
36,37
ε
codons at residues LC-Tyr92, LC-Thr93, or HC-Gly103 were
N -acetyl-(S)-lysine,
N -methyl-(S)-lysine,
and N -
Pyl
38
expressed in the presence of the AcrKRS/tRNA
pair
CUA
crotonyl-(S)-lysine (Kcr). To evolve a PylRS that can effi-
ciently charge tRNA
Pyl
(Figure 2A). In Terrific Broth (TB) media supplemented with
with 1, we generated a library based
CUA
5 mM 1, the yield of periplasmically secreted mutant proteins
on the sequence of the aaRS specific for Kcr, which differs
from 1 only by the presence of a single methyl group. Struc-
tural analysis of the PylRS active site indicated that mutations
at residues Leu266, Leu270, and Tyr271 might accommodate
the slightly less bulky acrylamide 1. Additionally, Leu274Ala
and Cys313Phe mutations are essential in reshaping the PylRS
active site to accommodate relatively small pyrrolysine ana-
logues, and mutation of Tyr349 to phenylalanine can improve
the PylRS amber suppression efficiency. Therefore, three resi-
dues (Leu266, Leu270, Tyr271) were varied by NNK random-
ization (N = A, G, T or C; K = G or T), and three fixed muta-
tions (Leu274Ala, Cys313Phe, Tyr349Phe) were introduced to
-
1
following purification over Protein G resin was ~1-2 mg L .
Electrospray ionization mass spectrometry (ESI-MS) con-
firmed independent incorporation of 1 at each of the three
suppressed sites (Figure 2A-C). Notably, the modified Fab’s
did not undergo any observable 1,4-addition reactions with
intracellular thiol nucleophiles, such as glutathione (GSH)
prior to secretion. An ErbB2 ELISA revealed that, while Her-
ceptin Fab-AcrK92 and Herceptin Fab-AcrK93 bound ErbB2
with an apparent K equivalent to the wild type (WT) Fab
d
(~0.4 nM), Herceptin Fab-AcrK103 binding was reduced al-
most 20-fold (Supplementary Information). Each of the mutant
proteins (6 ꢀM) was then incubated with 750 nM of the human
ErbB2 ECD (Met1-Thr652 with a C-terminal hexaHis tag) in
Dulbecco's Phosphate-Buffered Saline (DPBS), pH 8.8, for 48
h at 37 °C, followed by reducing SDS-PAGE to identify cova-
5
afford a library of 3×10 mutants (theoretical nucleic acid di-
4
versity of 3.3×10 ). Directed evolution was performed in E.
39
coli as previously described, resulting in identification of an
2
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Figure 2. Crosslinking reaction of genetically encoded acrylamide-bearing amino acids. (a-c) Mass spectrometry of 1 substituted in the
Herceptin Fab at LC-Tyr92 (a), LC-Thr93 (b), or HC-Gly103 (c) with expected masses of 47771, 47833, and 47877 Da, respectively. (d)
Reducing SDS-PAGE of 6 µM Herceptin Fab-AcrK mutants reacted with 750 nM ErbB2 ECD (residues Met1-Thr652 with a C-terminal
hexaHis tag) in DPBS, pH 8.8, for 48 h at 37 °C. The covalently crosslinked Herceptin Fab-ErbB2 ECD complex is indicated by an arrow-
head. (e) Reducing SDS-PAGE of Herceptin Fab-AcrF mutants with ErbB2 ECD under the same conditions as in (d).
lently crosslinked products. A new band was observed at ~125
kDa, which corresponds to a 1:1 covalent bond between the
ErbB2 ECD (apparent glycosylated mass ~100-110 kDa) and
reduced Herceptin Fab (~25 kDa). Densitometry revealed that
Assuming that periplasmic secretion would limit posttrans-
lational modification of 2 by GSH, Herceptin Fab mutants
with amber mutations replacing residues LC-Tyr92, LC-
Thr93, or HC-Gly103 were periplasmically expressed in the
Tyr
~
10% of the ErbB2 ECD reacted with the Herceptin Fab-
presence of the AcrFRS/tRNA
pair. Supplementation of
CUA
-1
AcrK93 mutant, while Herceptin Fab-AcrK92 and Herceptin
Fab-AcrK103 reacted only minimally (Figure 2D). Reducing
SDS-PAGE completely abrogated the noncovalent association
between the WT Fab and ErbB2 ECD (Supplementary Infor-
mation), indicating that 1 is responsible for the observed cross-
linking. These initial results suggest that a mutant Fab bearing
a genetically encoded Michael acceptor can form a covalent
complex with its native, unmodified binding partner. Howev-
er, the use of 1 required long incubation times (48 h) and non-
physiological pH (> 8.0), and afforded low crosslinking yields
TB media with 1 mM 2 typically yielded 4-6 mg L of mutant
protein following purification over Protein G resin. ESI-MS
confirmed successful incorporation of 2 without any observed
modification by cellular nucleophiles, and an ELISA con-
firmed binding of the mutants to ErbB2 with apparent K ’s
d
similar to the WT Fab (Supplementary Information). To assay
the formation of covalently crosslinked product, the Fab mu-
tants (6 ꢀM) were incubated with 750 nM ErbB2 ECD in
DPBS, pH 8.8, for 48 h at 37 °C, followed by reducing SDS-
PAGE. In contrast to the Herceptin Fab-AcrK mutants, in
which only mutation at LC-Thr93 resulted in crosslink for-
mation, crosslinking using 2 as the aza-Michael acceptor was
observed for the LC-Tyr92 mutant (66% reaction) and the HC-
Gly103 mutant (65% reaction) (Figure 2E). While the long
alkyl side chain of 1 may reach Lys569 from the LC-Thr93
site, 2 is structurally similar to the tyrosine it replaces at LC-
Tyr92 and is, therefore, better oriented for reaction with
Lys569. This optimal positioning, along with the increased
reactivity of the aryl acrylamide, may explain the increased
reaction efficiency observed using 2 for crosslinking to the
ErbB2 ECD.
(~10%).
Next we explored whether an alternative aza-Michael ac-
ceptor, p-acrylamido-(S)-phenylalanine (AcrF, 2), with less
side chain conformational flexibility and somewhat higher
2
5
reactivity due to the phenyl substituent, might lead to im-
proved crosslinking efficiency as compared to the lysine scaf-
fold. Synthesis of 2 was readily achieved in two steps with
90% overall yield (Supplementary Information). Many phe-
nylalanine-derived UAAs have been incorporated in response
to the amber nonsense codon using evolved Methanococcus
Tyr
jannaschii tyrosyl-tRNA synthetase (TyrRS)/tRNA
CUA
40
pairs. Additionally, certain evolved TyrRSs exhibit substrate
polyspecificity, wherein a single aaRS exhibits high permis-
sivity for UAAs, while preserving its ability to discriminate
Although acrylamides have been used successfully to cova-
lently modify adjacent cysteine residues, they are relatively
1
weak electrophiles and have not been particularly successful
41
13
against the 20 canonical amino acids. Using a fluorescence
for aza-Michael addition reactions at protein interfaces.
assay with a GFP-Tyr151TAG mutant, an existing polyspecif-
ic TyrRS42 (AcrFRS, mutations: Tyr32Val, Leu65Tyr,
Phe108His, Gln109Gly, Asp158Gly, Leu162Glu, Asp286Arg)
afforded a 2-fold fluorescence increase upon addition of 1 mM
Therefore, to further improve the yield of crosslinked product,
as well as the reaction conditions (time and pH), we designed
a novel optimized aza-Michael acceptor UAA with a vinyl-
sulfonamide group, p-vinylsulfonamido-(S)-phenylalanine
(VSF, 3). Vinylsulfonamides have recently been used to de-
2
. To confirm successful incorporation of 2, GFP-AcrF151
42
43
was expressed and purified from BL21(DE3) cells, followed
by characterization with SDS-PAGE and ESI-MS. The ob-
served mass (+302 Da) potentially corresponds to a GSH ad-
duct of GFP-AcrF151, which is likely due to its increased
electrophilicity compared to 1 (Supplementary Information).
sign inhibitors of ubiquitin activating enzymes and kinases,
and are somewhat more reactive than the corresponding
acrylamide moieties (reactivity: N-phenylacrylamide < N-
25
phenylvinylsulfonamide). Synthesis of 3 was achieved in
32% overall yield in two steps (Supplementary Information).
Because the polyspecific AcrFRS afforded only modest pro-
3
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Figure 3. Characterization of the reaction of Herceptin Fab-VSF92 with the ErbB2 ECD. (a) Mass spectrometry of 3 substituted in the
Herceptin Fab at LC-Tyr92 (Herceptin Fab-VSF92). The peak observed at 48149 Da likely represents a (+307 Da) GSH adduct. (b) Reduc-
ing SDS-PAGE of reactions of 4 µM Herceptin Fab-VSF92 with 1 µM ErbB2 ECD (residues Met1-Thr652 with a C-terminal hexaHis tag,
Sino Biological Inc., SBI) carried out for 2 h at 37 °C in DPBS at pH 6.4, 7.4, or 8.4. The covalently crosslinked Herceptin Fab-ErbB2
ECD complex is indicated by an arrowhead. (c) Reducing SDS-PAGE of time dependent reaction of 4 µM Herceptin Fab-VSF92 with 1
µM ErbB2 ECD (residues Met1–Thr652 with a C-terminal hexaHis tag, Sino Biological Inc., SBI) reacted in DPBS, pH 7.4, at 37 °C for
0
.2, 0.5, 1, 2, or 4 h. (d) Reducing SDS-PAGE of reaction of 5 ꢀM Herceptin fab-VSF92 with 1 µM ErbB2 ECD or ErbB2-Lys569Gln
ECD (residues Thr23–Thr630 with a C-terminal hexaHis tag expressed from HEK293 cells) in DPBS, pH 7.4, for 2 h at 37 °C.
tein yields of the GFP-VSF151 mutant in the presence of 1
tively. Furthermore, analyzing the time-dependence of the
reaction revealed covalently linked product after as short as 10
min, with ~96% conversion to crosslinked product achieved
within 4 h at pH 7.4 (Figure 3C). We additionally performed
the reaction in DPBS, pH 7.4 for 2 hours at 37 °C with 4 nM
Herceptin Fab-VSF92 and 1 nM ErbB2 ECD labeled with an
infrared dye in order to image the reaction product. Reducing
SDS-PAGE showed ~80% crosslinking using these concentra-
tions, which are similar to those used either in vitro or in vivo
(Supplementary Information). Thus, 3 is a highly efficient
crosslinker compared to 1 and 2, and forms covalent complex-
es at physiological pH. On the basis of the Herceptin Fab-
ErbB2 ECD co-crystal structure, we postulated that Herceptin
Fab-VSF92 is reacting with Lys569 of the ErbB2 ECD. To
test this hypothesis, the WT ErbB2 ECD and a Lys569Gln
mutant were expressed and purified from HEK293 cells. Each
ECD was incubated with Herceptin Fab-VSF92 at pH 7.4 for 2
h at 37 °C, followed by reducing SDS-PAGE (Figure 3D). A
covalent product was only observed with WT ErbB2 ECD,
while no reaction occurred between Herceptin Fab-VSF92 and
the Lys569Gln mutant. ELISA results confirmed that Her-
ceptin Fab-VSF92 bound to the ErbB2-Lys569Gln ECD with
an affinity indistinguishable from that of WT ErbB2 ECD
(Supplementary Information). Unfortunately, attempts to gen-
erate tryptic fragments of the crosslinked proteins that ionize
with appreciable efficiency were unsuccessful.
mM 3, we evolved an improved TyrRS that can more effi-
Tyr
ciently charge tRNA
with 3. A library was generated
CUA
using the AcrFRS sequence as a template, in which Val32,
Ile159, and Glu162 were varied by NNK mutagenesis to allow
for accommodation of the bulkier vinyl sulfonamide substitu-
ent, and Ala67 and His70 were varied using GST codons (S =
C or G, encoding Ala or Gly) to restrict steric bulk. A library
6
of 6×10 mutants was obtained (theoretical nucleic acid diver-
5
sity of 1.3×10 ), and directed evolution was performed in E.
3
9
coli as previously described. An aaRS (VSFRS, mutations:
Tyr32Gly, Leu65Tyr, Phe108His, Gln109Gly, Asp158Gly,
Ile159Leu, Leu162Gln, Asp286Arg) was identified that con-
tained three mutations to glycine residues, which enlarges the
active site and allows efficient incorporation of 3 into proteins.
Suppression of GFP-Asn149TAG in the presence of 1 mM 3
resulted in a 13-fold increase in fluorescence (Supplementary
Information). To confirm successful incorporation of 3 a GFP
mutant, GFP-VSF151, was expressed and purified from
BL21(DE3) cells, followed by characterization with ESI-MS.
The mutant protein was again observed as a (+303 Da) GSH
conjugate (Supplementary Information).
With a new aaRS in hand, Herceptin Fab-Tyr92TAG was
periplasmically expressed in the presence of the
Tyr
VSFRS/tRNA
pair. UAA 3 was incorporated at the same
CUA
site as 2, since they both similarly present the electrophilic
group on the phenylalanine scaffold. Supplementation of TB
Previous efforts to form covalent bonds between proteins
using genetically encoded UAAs required mutagenesis to in-
troduce a unique cysteine nucleophile into the target protein.
-
1
expression media with 1 mM 3 yielded ~4 mg L of mutant
protein following purification, and ESI-MS confirmed suc-
cessful incorporation (Figure 3A). A small portion of Her-
ceptin Fab-VSF92 is observed as a GSH conjugate (+307 Da)
in the mass spectrum, reflecting the enhanced reactivity of the
vinylsulfonamide group compared to the aryl acrylamide.
1
9
A novel feature of our covalent crosslinking methodology is
that the target protein does not require the introduction of a
nucleophilic residue and, as a consequence, may potentially be
crosslinked in its native cellular state. To test the biocompati-
bility of our crosslinking strategy with living cells, we first
confirmed that the reaction between Herceptin Fab-VSF92 and
the ErbB2 ECD is not affected by the presence of ~50% fetal
bovineserum (FBS). No off-target reactions with serum pro-
teins were observed by western blot using an anti-human kap-
pa light chains antibody (Supplementary Information). Next,
To explore the reactivity of this new Michael acceptor
UAA, we incubated 4 ꢀM Herceptin Fab-VSF92 with 1 ꢀM
ErbB2 ECD in DPBS adjusted to pH 6.4, 7.4, or 8.4 for 2
hours at 37 °C, followed by reducing SDS-PAGE (Figure 3B).
The reaction efficiency correlated with pH, with 53%, 86%,
and 95% crosslinking observed at pH 6.4, 7.4, and 8.4, respec-
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Figure 4. Specific and irreversible labeling of ErbB2 on the cell surface by Herceptin Fab-VSF92. (a) Herceptin Fab-VSF92 and Herceptin
Fab-WT (25 nM) were incubated with SK-BR-3 cells for 2 h at 37 °C, followed by total cell lysis and western blot analysis with an anti-
ErbB2 antibody. The covalently crosslinked Herceptin Fab-VSF92-ErbB2 complex is indicated by an arrowhead. (b) Coomassie-stained
reducing SDS-PAGE showing Herceptin Fab-WT and Herceptin Fab-VSF92 after non-specific labeling with FITC, and the corresponding
fluorescent image of the same gel. (c-h) Fluorescence imaging of live cells treated with Herceptin Fab(FITC)-WT or Herceptin Fab(FITC)-
VSF92. MDA-MB-468 and SK-BR-3 cells were cultured in DMEM supplemented with 10% FBS at 37 °C. At 70% confluence, 25 nM of
Herceptin Fab(FITC)-WT or Herceptin Fab(FITC)-VSF92 was added to the culture medium and incubated for 2 h in the dark. Green fluo-
rescent (left) and bright field (right) images were obtained under different wash conditions (the ‘gentle’ wash buffer was DPBS, and the
‘
stringent’ wash buffer was 100 mM glycine, pH 2.8, 300 mM NaCl, 3% tween 20) to show the irreversible labeling of cell surface ErbB2
with only Herceptin Fab(FITC)-VSF92 (h) but not with Herceptin Fab(FITC)-WT (g).
ErbB2-positive SK-BR-3 breast cancer cells cultured in Dul-
becco's Modified Eagle Medium (DMEM), pH 7.4, supple-
mented with 10% FBS were incubated with Herceptin Fab-
WT or Herceptin Fab-VSF92 (25 nM) for 0.5, 1, 2, or 12 h.
Western blot of a SDS-PAGE gel of the lysate with an anti-
ErbB2 antibody that binds to the intracellular domain of full
length ErbB2 revealed complete covalent labeling of ErbB2 by
Herceptin Fab-VSF92 within 2 h, whereas no covalent reac-
tion was observed for Herceptin Fab-WT (Figure 4A). We
next created fluorescent probes for live cell imaging by non-
specifically modifying the Herceptin Fab-WT and Herceptin
Fab-VSF92 with fluorescein isothiocyanate (FITC) to generate
Herceptin Fab(FITC)-WT and Herceptin Fab(FITC)-VSF92,
respectively (Figure 4B). ErbB2-positive SK-BR-3 cells and
ErbB2-negative MDA-MB-468 cells grown in DMEM, pH
both Herceptin Fab(FITC)-WT and Herceptin Fab(FITC)-
VSF92, while no fluorescence was observed on MDA-MB-
468 cells, confirming the selective labeling of ErbB2 (Figure
4C-F). To confirm the covalent nature of Herceptin
Fab(FITC)-VSF92 binding to cell-surface ErbB2, cells were
subjected to stringent washing conditions (100 mM glycine,
pH 2.8, 300 mM NaCl, 3% tween 20). The Herceptin
Fab(FITC)-VSF92 probe remained bound to ErbB2 at the cell
surface, while no fluorescence was observed on the cells pre-
viously labeled with Herceptin Fab(FITC)-WT (Figure 4G and
H). These results demonstrate that the genetically encoded
aza-Michael acceptor 3 can selectively and efficiently cova-
lently react with a proximal lysine nucleophile at the protein
binding interface both in vitro and in the presence of living
cells.
7
.4, with 10% FBS were incubated with Herceptin Fab(FITC)-
Conclusion
WT or Herceptin Fab(FITC)-VSF92 (25 nM) for 2 h. After
washing with DPBS to remove nonspecifically bound Fab, the
observed fluorescence with SK-BR-3 cells was similar for
Herein we have evolved orthogonal aaRS/tRNA pairs for
the genetic incorporation of a series of electrophilic UAAs, N -
acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine
ε
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(
AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF,
transfectin (Life Technologies) into FreeStyle HEK293 cells
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3). This series provides two different amino acid scaffolds,
lysine and phenylalanine, as well as incremental increases in
(Life Technologies) that were cultured to a density of 1×10
cells mL in 500 mL of FreeStyle 293 expression media
-
1
TM
reactivity
(reactivity:
N-alkylacrylamide
<
N-
(Life Technologies) at 37 °C and 5% CO . Expression media
2
2
5
phenylacrylamide < N-phenylvinylsulfonamide). We evalu-
ated these genetically encoded UAAs for covalently crosslink-
ing Herceptin Fab to ErbB2. While the acrylamide UAAs re-
sulted in modest crosslinking yields under basic conditions
containing secreted ErbB2 ECD was harvested and combined
every two days over the course of 6 days, followed by purifi-
cation using Ni-NTA agarose resin (Qiagen), with a yield of
-
1
~1-2 mg L . SDS-PAGE analysis showed that recombinant
ErbB2 ECD proteins migrated as the same molecular weight
as an equivalent ErbB2 ECD from a commercial source (Sup-
plementary Information). Characterization by mass spectrome-
try is not possible given the large molecular weight and exten-
sive glycosylation of these proteins.
(pH = 8.8), the vinylsulfonamide moiety resulted in rapid co-
valent crosslinking of the αErbB2-ErbB2 complex at physio-
logical pH with >95% yield, providing an extremely robust
protein-protein crosslinking strategy for native target proteins.
Because this strategy does not require genetic or chemical
modification of the target protein, it can potentially be directly
applied to cells or tissues. This methodology provides useful
covalent probes to identify protein interaction partners that
may be too weakly bound to be isolated by more conventional
affinity pull-down techniques. Moreover, covalent antibodies
of this type may be particularly useful for extracellular target-
ing applications, such as for diagnostics, imaging, and fluores-
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Crosslinking reactions. Reactions between Herceptin Fab
mutants (6 ꢀM) bearing 1 or 2 at LC-Tyr92, LC-Thr93, or HC-
Gly103 and 750 nM ErbB2 ECD were carried out in 20 ꢀL
DPBS (adjusted to pH 8.8 with bicine) at 37 °C for 48 h. Re-
actions between 1 ꢀM Herceptin Fab-VSF92 and 4 ꢀM ErbB2
ECD were carried out in 20 ꢀL DPBS, pH 7.4, at 37 °C for 2
h, unless otherwise specified. Prior to SDS-PAGE, reactions
were quenched by boiling at 90 °C in SDS-PAGE loading
buffer supplemented with β-mercaptoethanol for 5 min. Gel
band density was determined using ImageJ software
(http://rsbweb.nih.gov/ij/), and reaction completion was calcu-
lated as the intensity of the crosslinked product relative to total
ErbB2 (free plus crosslinked).
4
4,45
cence guided surgery.
We are currently determining the
generality of this approach by introducing electrophilic UAAs
into other therapeutic antibodies, protein-based protease inhib-
itors, growth factors, and cytokines. In addition, we are ex-
ploring the use of library based methods with affinity based
selections to identify productive crosslinking sites in protein
complexes for which detailed structural information is not
available.
Cell-based crosslinking of Herceptin Fab mutants was car-
5
ried out in a 12-well plate at a density of 1×10 cells per well
in DMEM supplemented with 10% FBS at 37 °C and 5% CO2.
At ~70% confluency, 25 nM Herceptin Fab-VSF92 or Her-
ceptin Fab-WT was added to each well and incubated for 0.5,
Experimental Section
Protocols for the chemical syntheses of UAAs 1, 2, and 3,
construction of library and expression plasmids, selections and
validation for the evolved aaRSs, and ELISA binding assays
are given in the Supplementary Information.
1
, 2, or 12 h. Cells were washed twice with cold DPBS and
lysed using 250 ꢀL of CelLytic M solution (Sigma) at 4 °C for
0 min. The clarified supernatant was analyzed by western
3
blot with an anti-human ErbB2 antibody (29D8, Cell Signal-
ing Technologies) that targets the intracellular domain of full
length ErbB2.
Protein expression and purification. Each pBAD-
Herceptin Fab-TAG (LC-Tyr92TAG, LC-Thr93TAG, or HC-
Gly103TAG) plasmid was co-transformed with pUltra-
AcrKRS/tRNA CUA, pUltra-AcrFRS/tRNA CUA, or pUltra-
VSFRS/tRNA
Pyl
Tyr
Fluorescent labeling of mammalian cells. Fluorescein
isothiocyanate (FITC, Molecular Probes) (770 ꢀM) was react-
ed with Herceptin Fab-VSF92 or Herceptin Fab-WT (58 ꢀM)
in 72 ꢀL of 200 mM sodium bicarbonate, pH 9.0, for 2 h at 25
Tyr
into E. coli TOP10 cells. Cells were cul-
CUA
-1
tured in TB media, supplemented with 100 ꢀg mL ampicillin,
-
1
5
0 ꢀg mL spectinomycin, and 5 mM 1, 1 mM 2, or 1 mM 3.
Protein expression was induced at an OD600 ~1.0 by addition
of 0.2% arabinose and mM isopropyl β-D-1-
thiogalactopyranoside (IPTG), and cells were cultured at 37
C (for proteins containing 1) or 25 °C (for proteins containing
or 3) for 16-24 h. Protein was released from pelleted cells by
periplasmic lysis with 20 mL lysis buffer (30 mM Tris-HCl,
°
C with shaking. Labeled protein was isolated from unreacted
dye using 0.5 mL Zeba spin desalting columns with a 40 kDa
MWCO (Pierce). The in-gel fluorescence image was acquired
on a Bio-Rad Gel Doc XR Imaging system with UV transillu-
mination. FITC-labeled Herceptin Fab-VSF92 or Herceptin
Fab-WT (25 nM) was added to ~70% confluent SK-BR-3 or
MDA-MB-468 cells (ATCC) cultured in DMEM supplement-
1
°
2
-
1
pH 7.5, 1 mM EDTA, 20% sucrose, 0.2 mg mL lysozyme)
per gram of wet cell pellet with shaking at 37 °C for 30 min.
Clarified lysate was applied to a 1 mL Protein G Sepharose 4
Fast Flow (GE Healthcare) column equilibrated with Buffer A
(50 mM NaOAc, pH 5.0), and Fab was eluted with Buffer B
(100 mM glycine, pH 2.8). Proteins were exchanged into
DPBS using centrifugal filtration with a 30 kDa molecular
weight cut-off (MWCO), followed by characterization with
ed with 10% FBS at 37 °C and 5% CO . After incubation in
2
the dark at 37 °C for 2 h, the cells were washed twice with
cold DPBS to remove unbound Fab. The SK-BR-3 cells were
then washed twice for 10 min at RT using either mild (DPBS)
or harsh (100 mM glycine, pH 2.8, 300 mM NaCl, 3% tween
2
0) conditions. Cells were imaged on a Nikon Eclipse Ti fluo-
rescence microscope with a 10x/0.30 Nikon Plan Fluor objec-
tive. FITC was excited with a Lumencor 64 mW light source
using a GFP/FITC 475/28 BP filter, and emission was detected
between 515 and 555 nm using an 8 sec exposure.
-
1
ESI-MS and SDS-PAGE. Yields were ~1-2 mg L for proteins
containing 1 and ~4-6 mg L for proteins containing 2 or 3.
-
1
Plasmids encoding the ErbB2 ECD (pFuse-ErbB2-His or
pFuse-ErbB2(Lys569Gln)-His ) were transfected using 293-
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Journal of the American Chemical Society
(
17) Huang, Y.; Wan, W.; Russell, W. K.; Pai, P.-J.; Wang, Z.;
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Supporting Information. Figures S1− S9, protein mass spectra,
Table S1, and detailed Materials and Methods. Correspondence
and requests for materials should be addressed to P.G.S. and
C.H.K. This material is available free of charge via the Internet at
http://pubs.acs.org.
(
19) Xiang, Z.; Ren, H.; Hu, Y. S.; Coin, I.; Wei, J.; Cang, H.;
Wang, L. Nat. Methods 2013, 10, 885.
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AUTHOR INFORMATION
Corresponding Authors
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schultz@scripps.edu
chkim@calibr.org
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Author Contributions
†
(
These authors contributed equally.
(
ACKNOWLEDGMENT
We thank Virginia Chambers for her assistance with manuscript
preparation. Research reported in this publication was supported
by the National Institutes of Health (NIH) under Award Number
F32GM099162 (J.L.F.) and by NIH R01 GM097206 (P.G.S.).
This is manuscript number 26045 of The Scripps Research Insti-
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