Genetically encoding fluorosulfate- l -tyrosine to react with lysine, histidine, and tyrosine via SuFEx in proteins in vivo

Article abstract of DOI:10.1021/jacs.8b01087

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics.

Full text of DOI:10.1021/jacs.8b01087

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Genetically encoding fluorosulfate-L-tyrosine to react with
lysine, histidine and tyrosine via SuFEx in proteins in vivo
Nanxi Wang, Bing Yang, Caiyun Fu, He Zhu, Feng Zheng, Tomonori Kobayashi,
Jun Liu, Shanshan Li, Cheng Ma, Peng George Wang, Qian Wang, and Lei Wang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b01087 • Publication Date (Web): 30 Mar 2018
Downloaded from http://pubs.acs.org on March 30, 2018
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Genetically encoding fluorosulfate-L-tyrosine to react with lysine,
histidine and tyrosine via SuFEx in proteins in vivo
‡
‡
‡
Nanxi Wang1 , Bing Yang1 , Caiyun Fu1,4 , He Zhu3, Feng Zheng2, Tomonori Kobayashi1, Jun Liu1, Shanshan
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Li1, Cheng Ma3, Peng G. Wang3, Qian Wang2, Lei Wang1*
1. Department of Pharmaceutical Chemistry and the Cardiovascular Research Institute, University of California San Francisco, 555
Mission Bay Blvd. South, San Francisco, California 94158, USA.
2. Hangzhou Research Institute of Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 310018, China.
3. Department of Chemistry and Center for Therapeutics and Diagnostics, Georgia State University, Atlanta, Georgia 30302, USA.
4. College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018, China.
Supporting Information Placeholder
within proteins to enhance photostability,2 increase thermo-
ABSTRACT: Introducing new chemical reactivity into pro-
teins in living cells would endow innovative covalent bond-
ing ability to proteins for research and engineering in vivo.
stability,3 staple proteins recombinantly,4 and build optical
nano-switches,5 and between proteins to pinpoint ligand-
receptor interaction,6 target native receptors irreversibly,4,7
Latent bioreactive unnatural amino acids (Uaas) can be in-
generate covalent macromolecular inhibitors,8 and capture
corporated into proteins to react with target natural amino
weak protein-protein interactions.9 Bioreactive Uaas have
acid residues via proximity-enabled reactivity. To expand the
initially been developed to target Cys2 and subsequently to
diversity of proteins amenable to such reactivity in vivo, a
chemical functionality that is biocompatible and able to react
with multiple natural residues under physiological conditions
target Lys, His and Tyr.3-4,7,10 Reacting with Lys, His and Tyr
often requires basic conditions that are infeasible in vivo.4,10b
At this burgeoning stage, most genetically encoded bioreac-
tive Uaas react with 1 to 2 natural residues only, have modest
to severe cytotoxicity, and are primarily used for in vitro ap-
plications.
is highly desirable. Here we report the genetic encoding of
fluorosulfate-L-tyrosine (FSY), the first latent bioreactive
Uaa that undergoes sulfur-fluoride exchange (SuFEx) on
proteins in vivo. FSY was found nontoxic to E. coli and mam-
malian cells; after being incorporated into proteins, it selec-
tively reacted with proximal lysine, histidine, and tyrosine via
SuFEx, generating covalent intra-protein bridge and inter-
protein crosslink of interacting proteins directly in living
cells. The proximity-activatable reactivity, multi-targeting
ability, and excellent biocompatibility of FSY will be invalua-
ble for covalent manipulation of proteins in vivo. Moreover,
genetically encoded FSY hereby empowers general proteins
with the next generation of click chemistry, SuFEx, which
will afford broad utilities in chemical biology, drug discovery,
and biotherapeutics.
An ideal chemical functionality for latent bioreactive Uaas
should be stable, nontoxic, and nonreactive inside cells, yet
upon placed in proximity to target residues becomes reactive
under cellular conditions. Aryl fluorosulfates are weak elec-
trophiles attracting great attention for the new generation of
click chemistry:11 they are relatively unreactive toward hy-
drolysis, thermolysis and reduction,11-12 and are unreactive
toward free amino acids under physiological conditions,13
but form covalent bonds with Tyr, Lys and Ser through sul-
fur-fluoride exchange (SuFEx) reaction when used as war-
heads in chemical probes specifically binding proteins,14 alt-
hough a positively charged microenvironment is required for
reactivity. Given the tremendous potential of aryl fluorosul-
fate for biocompatible reactivity emerging from recent small
molecule studies, it is desirable to genetically install this func-
tional group onto proteins so that its unique properties can
be exploited in large biomolecules and in vivo.
The ability to endow proteins with new chemical reactivity
will expand novel avenues for protein research and engineer-
ing, potentially impacting biological studies, biotherapeutics,
and synthetic biology.1 The genetic code has recently been
engineered to encode a new class of unnatural amino acids
(Uaas), the latent bioreactive Uaas.2 These Uaas, after incor-
poration into proteins, react with a target natural amino acid
residue via proximity-enabled reactivity, enabling selective
formation of new covalent linkages within and between pro-
teins. The new covalent bonding ability has been harnessed
Here we evolved a new tRNA-synthetase pair to genetical-
ly incorporate fluorosulfate-L-tyrosine (FSY, Fig. 1a) into
proteins in E. coli and mammalian cells. We found that FSY
was generally nontoxic to cells, and was able to react with
Lys, His, and Tyr specifically via proximity-enabled SuFEx
reaction within and between proteins under physiological
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conditions (Fig. 1b). We demonstrated the crosslinking of
interacting proteins using FSY directly in vivo.
TAG codon at position 36 (Afb-36TAG) was co-expressed
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()*
with the tRNA
/FSYRS pair in E. coli. In the absence of
%&'
FSY, no full-length Afb was detected; when 1 mM FSY was
added in growth media, full-length Afb36FSY was produced
with a yield of 1.6 mg/L (Fig. 1c). The purified Afb36FSY
was analyzed by electrospray ionization time-of-flight mass
spectrometry (ESI-TOF MS) (Fig. 1d). A peak observed at
7855.96 Da corresponds to intact Afb containing FSY at site
36 (Afb36FSY: expected 7856.69 Da). A peak measured at
7724.77 Da corresponds to Afb36FSY lacking the initiating
Met (Afb36FSY-Met: expected 7725.50 Da). Two minor
peaks observed at 7836.55 and 7705.16 Da correspond to
Afb36FSY lacking F (expected 7836.69 Da) and Afb36FSY-
Met lacking F (expected 7705.49 Da), respectively, suggest-
ing slight F elimination during MS measurement. Notably,
no peaks corresponding to Afb containing other amino acids
at position 36 were observed. We also incorporated FSY at
position 24 of the Z protein and analyzed with tandem MS. A
series of b and y ions unambiguously indicate that FSY was
incorporated at the TAG-specified position 24 (Fig. 1e). The
presence of 1 mM FSY did not affect E. coli growth (Fig. S1),
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suggesting no obvious cytotoxicity. These results indicate
()*
%&'
that the evolved tRNA
/FSYRS pair was able to incorpo-
rate FSY with high efficiency and specificity in E. coli.
We next tested FSY incorporation into proteins in mam-
malian cells. We transfected HeLa-EGFP-182TAG reporter
cells18 with plasmid pMP-FSYRS-3xtRNA, which expresses
()*
%&'
FSYRS and tRNA
genes. Suppression of the 182TAG
codon of the genome-integrated EGFP gene would produce
full-length EGFP rendering cells fluorescent. Strong EGFP
fluorescence was measured from cells by flow cytometry only
when FSY was added (Fig. 2a). The fluorescence intensity
increased with FSY concentration and incubation time (Fig.
2b). As a positive control, we incorporated p-azido-L-
phenylalanine (AzF) into reporter cells in parallel using
plasmid pIre-Azi3 (Fig. S2), which is the most efficient Uaa
incorporation system in mammalian cells in our hands.6 FSY
incorporation reached 76% of the AzF level. Notably, while
cellular toxicity is often an issue with bioreactive Uaas, we
did not observe obvious toxicity of FSY to HeLa or
HEK293T cells (Fig. S3), a valuable property of FSY possi-
bly owing to the extremely low background reactivity of aryl
fluorosulfate inside cells.14a The above results were also con-
firmed by fluorescence confocal microscopy (Fig. 2c). In the
presence of FSY, strong EGFP fluorescence was observed
throughout the cells, and cell morphology remained normal.
No fluorescence signal was detected when FSY was not add-
ed. We also incorporated FSY into proliferating cell nuclear
Figure 1. Genetically encode FSY into proteins in E. coli. (a)
Structure of FSY. (b) Scheme showing proximity-enabled
SuFEx reaction between FSY and a natural nucleophilic residue.
(c) SDS-PAGE showing FSY incorporation into Afb(36TAG)
in E. coli. (d) ESI-TOF MS spectrum of intact Afb-36FSY. (e)
Tandem MS spectrum of Z-24FSY.
FSY was synthesized using the SO2F2/borax method (88%
yield, Supplementary methods).11,15 To genetically encode
FSY, we evolved a mutant pyrrolysyl-tRNA synthetase
(PylRS) specific for FSY. A PylRS mutant library was gener-
ated by mutating residues Ala302, Leu305, Tyr306, Leu309,
Ile322, Asn346, Cys348, Tyr384, Val401, and Trp417 of the
Methanosarcina mazei PylRS using the small-intelligent mu-
tagenesis approach,16 and subjected to selection as de-
scribed.17 Six hits showing FSY-dependent phenotype were
identified; they all converged on the same amino acid se-
quence (302I/346T/348I/384L/417K), which we named as
FSYRS.
antigen (PCNA) through transient transfection of PCNA-
()*
%&'
165TAG and tRNA
/FSYRS genes into HEK293T cells.
Full-length PCNA was produced in the presence of FSY, with
incorporation efficiency reaching 41% of AzF (Fig. 2d). The-
se results suggest that FSY was incorporated into proteins in
We evaluated the incorporation specificity of FSY into pro-
teins in E. coli. The Zspa affibody (Afb) gene containing a
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mammalian cells with high efficiency and specificity without weight corresponding to MBP-Z24FSY and Afb adducts
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causing detrimental effects.
(Fig. 3b). We then purified 6xHis-tagged proteins from cells
and analyzed with SDS-PAGE. Consistently, a protein band
corresponding to the crosslinked MBP-Z with Afb was clear-
ly observed for Afb-7Lys, Afb-7His, and Afb-7Tyr (Fig. 3b),
with crosslinking efficiency (determined by band intensities)
of 59%, 53% and 35%, respectively. In contrast, no cross-
linking bands were observed when MBP-Z24FSY was co-
expressed with Afb-7Cys, Afb-7Ser, Afb-7Thr, or Afb-7Ala.
While aryl carbamate requires basic pH to crosslink Lys or
Tyr at Afb/Z interface in vitro,10b FSY was able to crosslink
Lys, His or Tyr directly in live E. coli cells.
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Figure 2. Genetically encode FSY into proteins in mammalian
cells. (a) FACS analysis of FSY incorporation into EGFP-
182TAG in HeLa cells. (b) Total EGFP fluorescence intensity
measured from the same number of HeLa-EGFP-182TAG re-
porter cells. Error bar: s.e.m., n = 6. (c) Fluorescence images of
HeLa-EGFP-182TAG reporter cells. (d) Western blot analysis
of FSY and AzF incorporation into PCNA-165TAG in
HEK293T cells. An anti-His antibody was used to detect the
6xHis-tag appended at the C-terminus of PCNA.
We then determined whether the incorporated FSY could
react with natural amino acid residues via proximity-enabled
reactivity directly in E. coli. Afb binds to its substrate Z pro-
tein with a moderate affinity, providing a suitable protein
framework to study FSY crosslinking in vivo. In light of the
crystal structure of Afb-Z complex,19 we introduced FSY at
position 24 of Z protein and the target natural residue at po-
sition 7 of Afb, placing the two residues in close proximity
upon Afb-Z binding (Fig. 3a). As aryl fluorosulfate is a weak
electrophile, we decided to test FSY’s reactivity toward Lys,
His, Tyr, Cys, Ser, and Thr using Ala as a negative control. To
better separate the Afb and Z proteins of similar molecular
weights, we fused maltose binding protein (MBP) to the N-
terminus of Z (MBP-Z). MBP-Z and Afb were both append-
ed with a 6xHis-tag at C-terminus. To determine whether
chemical crosslinking could occur in living cells, we co-
expressed MBP-Z24FSY and Afb-7X (X = target residue) in
E. coli. After culturing at 37 °C for 6 h, the same number of
cells were analyzed using Western blot under denatured con-
ditions. From cells expressing Afb-7Lys, Afb-7His, or Afb-
7Tyr, crosslinking bands were observed with molecular
Figure 3. FSY crosslinks proximal Lys, His and Tyr via SuFEx
directly in E. coli cells. (a) Structure of Afb-Z complex showing
two proximal sites for FSY and target residue X incorporation.
(b) Top: Western blot of E. coli cell lysates; Bottom: SDS-PAGE
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interacts with PAPS reductase to facilitate the reduction of
of proteins His-tag purified from E. coli. (c-e) Tandem MS spec-
trum of MBP-Z-24FSY/Afb-7Lys (c), MBP-Z-24FSY/Afb-7His
(d) and MBP-Z-24FSY/Afb-7Tyr (e).
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adenylated sulfate to sulfite for de novo cysteine biosynthe-
sis.20 On the basis of the complex structure of PAPS reduc-
tase with Trx1,20 we incorporated FSY into E. coli Trx1 at site
62 to target the proximal Tyr191 of PAPS reductase (Fig.
5a). We expressed and purified Trx1(62FSY) and WT PAPS
reductase, and incubated them in Tris buffer at pH 7.4 or 8.0
for 12 h. SDS-PAGE showed clear bands corresponding to
the covalent complex of Trx1 with PAPS reductase (Fig. 5b,
Fig. S4). Tandem MS analysis of the sample unambiguously
indicate that FSY of Trx1 covalently crosslinked with the
target Tyr191 of PAPS reductase via SuFEx reaction (Fig.
5c). Taken together, the intramolecular CaM crosslinking
and intermolecular Trx1-PAPS reductase crosslinking further
corroborate that FSY reacted with Tyr in proximity via
SuFEx reaction.
To further validate the in vivo chemical crosslinking ability
of FSY, we analyzed the purified proteins using tandem MS.
As expected, strong signals corresponding to the covalently-
linked peptides of MBP-Z24FSY and Afb-7Lys were identi-
fied (Fig. 3c). A series of b and y fragmented ions clearly in-
dicate that the incorporated FSY crosslinked exclusively with
Lys 7 of Afb. Similar MS results were also obtained for MBP-
Z24FSY co-expressed with Afb-7His or Afb-7Tyr, confirm-
ing FSY crosslinked with the target His7 (Fig. 3d) or Tyr7
(Fig. 3e), respectively. Meanwhile, consistent with Western
and SDS-PAGE results, no crosslinked peptides of MBP-
Z24FSY with Afb-7Ser, Afb-7Thr, Afb-7Cys, or Afb-7Ala
were detected by tandem MS.
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In addition, we incorporated FSY and Tyr into a single
protein to test intramolecular crosslinking in vivo. We co-
()*
%&'
expressed in E. coli the tRNA
/FSYRS pair with a mutant
calmodulin gene (CaM-76TAG), which encoded 76TAG for
FSY incorporation and Tyr at a nearby site 80 (Fig. 4a). This
CaM protein was expressed in the presence of 1 mM FSY,
purified (Fig. 4b), and analyzed with tandem MS (Fig. 4c). A
series of b and y fragment ions unambiguously show that FSY
formed a covalent linkage with Tyr80 via SuFEx.
Figure 5. FSY crosslinks Tyr via SuFEx intermolecularly. (a)
Structure of Trx1 in complex with PAPS reductase showing FSY
site and the native Tyr191. (b) SDS-PAGE and (c) tandem MS
spectrum of Trx1 crosslinked with PAPS reductase.
In summary, we genetically encoded the live-cell friendly
FSY into proteins in E. coli and mammalian cells, which selec-
tively reacted with proximal Lys, His and Tyr residues via
SuFEx directly in live E. coli cells. Intermolecular crosslinking
using bioreactive Uaas has been mainly limited to in vitro
usage with few exceptions targeting Cys;6,9a FSY enables in-
termolecular crosslinking of interacting proteins in vivo and
targeting three different residues. Since FSY’s target residues
are often found at protein surface and interface, FSY will
dramatically expand the diversity of proteins amenable to
covalent bonding and enable creative in vivo applications to
exploit protein covalent bonding ability. Moreover, genetical-
ly encoding FSY now empowers proteins with the new gen-
Figure 4. FSY crosslinks Tyr via SuFEx intramolecularly in E.
coli cells. (a) Structure of CaM showing sites for FSY and target
Tyr. (b) SDS-PAGE and (c) tandem MS spectrum of purified
CaM-76FSY-80Tyr.
To demonstrate that FSY could crosslink interacting pro-
teins through targeting a native Tyr residue, we tested wheth-
er FSY-armed thioredoxin (Trx) could covalently capture 3′-
phosphoadenosine-5′-phosphosulfate (PAPS) reductase. Trx
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Hett, E. C.; Kyne, R. E., Jr.; Robinson, R. P.; Jones, L. H. ACS Chem. Biol.
2017, 12, 2015-2020.
(15) Chen, W.; Dong, J.; Li, S.; Liu, Y.; Wang, Y.; Yoon, L.; Wu, P.;
Sharpless, K. B.; Kelly, J. W. Angew. Chem. Int. Ed. Engl. 2016, 55, 1835-
1838.
eration of click chemistry, SuFEx, which will find broad ap-
plications in chemical biology, drug discovery, and biothera-
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peutics.
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ASSOCIATED CONTENT
(16) Lacey, V. K.; Louie, G. V.; Noel, J. P.; Wang, L. ChemBioChem
2013, 14, 2100-2105.
(17) (a) Wang, L.; Schultz, P. G. Angew. Chem. Int. Ed. Engl. 2005, 44,
34-66. (b) Takimoto, J. K.; Dellas, N.; Noel, J. P.; Wang, L. ACS Chem. Biol.
2011, 6, 733-743.
(18) Wang, W.; Takimoto, J. K.; Louie, G. V.; Baiga, T. J.; Noel, J. P.;
Lee, K. F.; Slesinger, P. A.; Wang, L. Nat. Neurosci. 2007, 10, 1063-1072.
(19) Hogbom, M.; Eklund, M.; Nygren, P. A.; Nordlund, P. Proc. Natl.
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Supporting Information. The Supporting Information is avail-
able free of charge on the ACS Publications website.
Experimental details, Figure S1-S3 (PDF)
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AUTHOR INFORMATION
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Corresponding Author
(20) Chartron, J.; Shiau, C.; Stout, C. D.; Carroll, K. S. Biochemistry
2007, 46, 3942-3951.
* Lei.Wang2@ucsf.edu
Author Contributions
‡These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
L.W. acknowledges the support of the NIH (R01GM118384,
RF1MH114079).
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210. (c) Fadeyi, O. O.; Hoth, L. R.; Choi, C.; Feng, X.; Gopalsamy, A.;
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Figure 1
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ACS Paragon Plus Environment

Journal of the American Chemical Society
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Figure 2
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Figure 3
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ACS Paragon Plus Environment

Journal of the American Chemical Society
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Figure 5
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ACS Paragon Plus Environment

Journal of the American Chemical Society
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