A genetically encoded 19F NMR probe for tyrosine phosphorylation

Article abstract of DOI:10.1002/anie.201300463

Simple and selective: Tyrosine phosphorylation is a pivotal post-translational modification which regulates the enzymatic activity, protein conformation, and protein-protein interactions. The highly efficient genetic incorporation of 3,5-difluorotyrosine (F2Y) in E. coli and the use of F2Y as a ¹⁹F NMR probe for the tyrosine phosphorylation are reported (see picture).

Full text of DOI:10.1002/anie.201300463

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DOI: 10.1002/anie.201300463
Protein–Protein Interactions
A Genetically Encoded 19F NMR Probe for Tyrosine
Phosphorylation**
Fahui Li, Pan Shi, Jiasong Li, Fan Yang, Tianyuan Wang, Wei Zhang, Feng Gao, Wei Ding,
Dong Li, Juan Li, Ying Xiong, Jinpeng Sun, Weimin Gong,* Changlin Tian,* and
Jiangyun Wang*
Dedicated to Professor Yufen Zhao on the occasion of her 65th birthday
Tyrosine phosphorylation is a pivotal post-translational
modification (PTM) which regulates enzymatic activity,
protein conformation, and protein–protein interactions.[1]
While the eukaryotic protein tyrosine kinases (PTKs) have
been intensively studied in the past three decades because of
their great importance in cellular signaling and diseases,[2] the
prokaryotic PTKs, which play ubiquitous roles in bacterial
virulence, were found only recently, and their activation
mechanism is controversial.[3]
fluorinated amino acids into proteins.[5] Among these meth-
ods, the genetic code expansion technique has the unique
advantages that fluorinated amino acids can be directly
incorporated into specific sites of any protein of interest in
living cells, and that the mutant protein can be easily obtained
in milligram quantities. However, this powerful method has
not been applied to investigate tyrosine phosphorylation.
Here we report the highly efficient genetic incorporation
of the unnatural amino acid (UAA) 3,5-difluorotyrosine
1 (hereafter termed F2Y, Scheme 1), which mimics the
19F NMR spectroscopy has recently emerged as a powerful
tool for characterizing enzyme mechanisms and protein
motions over a range of time scales, because of the high
intrinsic sensitivity of fluorine, 100% natural abundance of
the NMR-active isotope, the absence of any natural back-
ground in proteins, and the exquisite sensitivity of the 19F
chemical shift to environment.[4] Numerous chemical and
biosynthetic methods have been developed for incorporating
[*] P. Shi,[+] J. S. Li,[+] D. Li, J. Li, Y. Xiong, C. L. Tian
School of Life Sciences, University of Science and
Technology of China, Hefei, Anhui, 230026 (China)
E-mail: cltian@ustc.edu.cn
Scheme 1. Reversible tyrosine (A) or F2Y (B) phosphorylation and
dephosphorylation catalyzed by PTK and PTP, respectively.
F. H. Li,[+] J. S. Li,[+] T. Y. Wang, W. Zhang, F. Gao, W. Ding,
W. M. Gong, J. Y. Wang
Laboratory of Non-coding RNA, Institute of Biophysics
Chinese Academy of Sciences
15 Datun Road, Chaoyang District
Beijing, 100101 (China)
tyrosine substrate, into the activation loops (a-loop) of the
bacterial PTK Etk and eukaryotic PTK Src in E. coli. While
the presence of phosphotyrosine (pTyr) in the a-loop of many
PTKs is well established, its exact role in regulating PTK
activity and interacting with drug molecules is still not fully
understood. A major limitation of our understanding is that
such a PTM cannot be directly probed through traditional
site-directed mutagenesis methods, and that pTyr is often
susceptible to hydrolysis during the protein crystallization
process. As a result, it remains very difficult to obtain
structural information to elucidate how pTyr interacts with
other residues or drug molecules. Through the genetic
incorporation of 1 and using 19F NMR spectroscopy, we
provide for the first time direct evidence that Arg614 interacts
with the a-loop residue pTyr574 to lock Etk in an active
conformation. Substitution of Arg614 to Ala does not impair
the autophosphorylation activity of Etk on residue Tyr574,
but results in an inactive conformation with significantly
decreased overall activity. We also show that dasatinib (a
cancer drug targeting BCR/ABL and Src) binding to Src
causes a significant conformation change in its a-loop bearing
E-mail: wgong@ibp.ac.cn
jwang@ibp.ac.cn
F. Yang, J. P. Sun
Department of Physiology
Shandong University School of Medicine
Jinan 250012 (China)
P. Shi,[+] C. L. Tian
High Magnetic Field Laboratory
Chinese Academy of Sciences
Hefei, Anhui, 230031 (China)
[+] These authors contributed equally to this work.
[**] We gratefully acknowledge the Major State Basic Research Program
of China (grant numbers 2010CB912301 and 2009CB825505), the
National Science Foundation of China (grant numbers 90913022,
31000364, 31170817, and 31100563), the CAS grant (grant number
KSCX2-EW-G-7) to J.W., F.L., C.T., and Y.X. We thank Professors
Zongchao Jia and Jingxuan Pan for helpful discussion.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201300463.
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pTyr416. These insights cannot be obtained by conventional
methods including X-ray diffraction (XRD) or mass spec-
trometry (MS).[3] Our new method should be broadly
applicable to the investigation of the tyrosine phosphoryla-
tion mechanism, protein–protein interactions mediated by
tyrosine phosphorylation, and drug–PTK interactions.
Our investigation started from the synthesis of F2Y 1 and
o-phospho-3,5-difluorotyrosine (pF2Y), following established
methods.[1b,5l] The UAA F2Y was selected as a surrogate
substrate of PTK, since it has been previously demonstrated
that peptides containing F2Y showed similar efficiency as
a PTK substrate compared with the corresponding tyrosine-
containing peptide.[6] We found that the 19F chemical shifts of
F2Y and pF2Y were at d = À133.1 ppm and d = À126.7 ppm,
respectively (see Figure S1 in the Supporting Information),
which allowed for convenient differentiation of the F2Y and
pF2Y 19F NMR signals.
To selectively incorporate F2Yat defined sites in proteins,
a
mutant M. jannaschii tyrosyl amber suppressor
tRNA(MjtRNATyrCUA)/tyrosyl-tRNA synthetase (MjTyrRS)
pair was evolved that uniquely specifies 1 in response to the
TAG codon, as previously reported.[5j,k] One MjTyrRS clone
emerged after positive–negative selections, which grew at
160 mgmLÀ1 of chloramphenicol in the presence of 1 mm 1,
but only at 20 mgmLÀ1 chloramphenicol in its absence, and
was named F2YRS (Tyr32Arg, Leu65Tyr, His70Gly,
Phe108Asn, Gln109Cys, Asp158Asn, Leu162Ser).
Figure 1. a) Structure of F2YRS with F2Y bound in the active site; the
To unravel the structural basis for the selective recogni-
tion of F2Y by F2YRS, we solved the crystal structures of
F2YRS, in the presence and absence of F2Y. Since the phenol
group of the F2Y substrate has a pKa of 6.8,[5e] F2Y is likely to
be present in an anionic state, and binds favorably to the
positively charged residue Arg32. One of the guanidine
nitrogen atoms of Arg32 is involved in hydrogen-bonding
interactions with the phenolic oxygen atom of F2Y. The
amide nitrogen atom of Asn158 is located 3.4 ꢀ away from
one of the fluorine atoms in F2Y, and one of the terminal
guanidine nitrogen atoms of Arg32 is situated 3.5 ꢀ away
from the other fluorine atom in F2Y (Figure 1A). While these
distances are too far away for hydrogen bonding, relatively
strong dipolar interactions between the fluorine atoms and
amide/guanidine groups may be present.[7] Our observation is
consistent with previous studies that organic fluorine is only
a weak hydrogen-bond acceptor.[8] We then superimposed the
apo and holo F2YRS structures. As shown in Figure 1B, most
of the side-chain atoms in the F2YRS-active site make only
small movements (less than 1 ꢀ) to accommodate the F2Y
substrate, which indicates a pre-organized active site. This
feature may be important for maximal substrate binding
because of the minimization of entropic loss, and the high
catalytic efficiency of F2YRS. To gain further insight on how
F2YRS discriminates against the tyrosine substrate, we
performed structure alignment of F2YRS and the wt
MjTyrRS. As shown in Figure S2, the backbone of F2YRS
and MjTyrRS can be superimposed, and the tyrosine ligand
occupies a similar position as F2Y. If F2YRS binds a tyrosine
in its active site, the tyrosine ligand will displace some water
molecules, decrease the local dielectric constant, and increase
the free-energy of the positively charged residue Arg32. Since
Fo-Fc electron density map of F2Y was contoured at 2.7 s. b) Super-
position of the active sites of holo F2YRS (green) and apo F2YRS
(blue). Nitrogen atoms are shown in blue, oxygen atoms are shown in
red, and fluorine atoms are shown in cyan.
the tyrosine ligand is likely to be present in a charge-neutral
state, as the pKa of the phenol group is 10.2, the ligand will
not be able to stabilize the positive charge of Arg32 as F2Y
does.
To determine whether F2YRS can facilitate the incorpo-
ration of F2Y into proteins with high efficiency and fidelity,
we performed in vitro amino-acylation assays. As Figure 2A
shows, F2YRS only charged F2Y, but not tyrosine, to
tRNATyrCUA. The F2YRS was then cloned into pEVOL
plasmid as described[5k] for enhanced expression of proteins
containing F2Y. An amber stop codon was substituted for
Tyr151 in the green fluorescent protein (GFP). Protein
production was carried out in E. coli in the presence of
F2YRS, MjtRNATyrCUA, and 0.5 mm F2Y, or in the absence of
F2Y as a negative control. Analysis of the purified GFP-151-
F2Y by SDS-PAGE showed that full-length GFP was
expressed only in the presence of F2Y (Figure 2B), indicating
that F2YRS was specifically active for F2Y but inactive for
any natural amino acids. ESI-MS analysis of the GFP-151-
F2Y mutant gave an observed average mass of 27746.0 Da, in
agreement with the calculated mass of 27746.0 Da (Figure 2C
and D). The yield for mutant GFP was 60 mgLÀ1. For
comparison, the yield of wild-type GFP was 100 mgLÀ1. This
corresponded to a UAG codon suppression efficiency of 60%.
To the best of our knowledge, this is the highest UAG codon
suppression efficiency ever reported for UAA incorporation.
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the yield of wt Etk was
20 mgLÀ1 (Figure 4A). Tryptic
digestion and tandem mass
spectrometry confirmed that
F2Y was selectively incorpo-
rated in the 574 position (Fig-
ure S3). Etk-574-F2Y auto-
phosphorylation or dephos-
phorylation was then quantified
using 19F solid-state NMR spec-
troscopy[11] and western blot
assays. An aliquot of each
sample was saved for western
blot analysis, and the remaining
samples were frozen at À408C,
lyophilized, and subjected to
solid-state NMR spectra acquis-
ition. As shown in Figure 4C,
the Etk-574-F2Y sample which
was allowed to auto-phosphor-
ylate gave rise to two 19F NMR
signals at À122.3 ppm and
À134.5 ppm,
respectively.
Upon PTP1B treatment, how-
ever, the peak at À122.3 ppm
disappeared, whereas the peak
at À134.5 ppm remained intact
(Figure S4). These results indi-
Figure 2. A) In vitro amino-acylation of tRNATyrCUA by F2YRS. B) Coomassie-stained SDS-PAGE of TAG151
mutant GFP (indicated by a black arrow) expression in the presence and absence of 0.5 mm F2Y. C) ESI-
MS spectra of the GFP-TAG151-F2Y mutant. D) Deconvoluted spectrum. Expected mass: 27746 Da,
found: 27746 Da.
This high efficiency is essential for NMR experiments since
a large amount of sample is required, and eukaryotic PTK can
be produced with only relatively low yield in E. coli.[3g]
Etk is an inner-membrane PTK in gram-negative bacteria,
which plays vital roles in regulating the polymerization and
transport of virulence-determining capsular polysaccharide
(CPS).[2] The cytoplasmic domain of E. coli Etk (residues 451–
726) contains a conserved Tyr574 in its a-loop. Through
structural and mutational analysis, it was proposed that Etk
activation requires Tyr574 auto-phosphorylation. The a-loop
phosphorylation is also required for the activation of many
eukaryotic PTK.[9] As shown in Figure 3, in the inactive state,
the Tyr574 side chain is situated in close proximity to the ADP
substrate, blocking the substrate peptide from accessing the
Etk active site. Once Tyr574 is phosphorylated, previously
reported structural models suggested that it moves away from
the catalytic center, binds to Arg614, and adopts a conforma-
tion that allows for substrate binding and catalysis.[3g] How-
ever, phosphorylated Tyr574 was not observed in a recently
reported crystal structure,[3g] because of rapid dephosphor-
ylation in the XRD sample preparation process.[2] Indeed, it is
well known that pTyr is less stable in phosphoamino acid
analysis than pSer/pThr in general.[10]
To verify if the a-loop Tyr574 is indeed phosphorylated as
previously proposed, we expressed the Etk C-terminal
cytoplasmic domain (residues 451–726) bearing F2Y at
position 574 (termed Etk-574-F2Y) in E. coli, using
pEVOL-F2YRS and pET28c-Etk574TAG plasmids. The
yield of Etk-574-F2Y mutant was 12.5 mgLÀ1. In comparison,
Figure 3. Left: Structure of E coli PTK Etk (pdb code 3CIO). Right:
Structure model of Etk, bearing a pTyr574 in its a-loop.
cate that the peak at À122.3 ppm corresponds to pF2Y at
position 574. Consistent with these results, the pTyr signals in
Etk-574-F2Y disappeared after PTP1B treatment, in a west-
ern-blot assay using the anti-pTyr antibody. Notably, the
NMR signal intensity of pF2Y was only 3.8% in comparison
to that of F2Y, indicating that only a small fraction of tyrosine
was phosphorylated in the a-loop of Etk. These results are
consistent with previous reports that phosphorylation of the
a-loop tyrosine in bacterial PTK is very low but sufficient to
elicit extensive inter-phosphorylation.[3e] Based on the Etk
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signal of the unphosphorylated protein. This is due to
different conformational flexibility of the two forms.
Since there is currently strong interest in using 19F NMR
spectroscopy to investigate drug–protein interactions,[4] we
then verified if our method is applicable for detecting the
interaction between dasatinib and c-Src, a key enzyme in
cancer formation.[14,3j–l] Src catalytic activity requires phos-
phorylation of Tyr416 in the a-loop. If Tyr416 is not
phosphorylated, the a-loop adopts an alpha-helical, ordered
but inhibitory conformation (Figure S6), blocking the sub-
strate-binding site. Phosphorylation of Tyr416 stabilizes the a-
loop far away from the active site, opening it up for catalytic
turnover.[3i–n] While a complex structure of dasatinib and c-Src
was recently reported,[3k] its a-loop appeared disordered
(Figure S6), and it is not known if dasatinib binding to c-Src
can cause conformation changes in its a-loop. To directly test
this, we expressed the chicken c-Src bearing F2Y at position
416 (termed Src-416-F2Y), as previously described.[3n] The
conformation change of Src-416-F2Y auto-phosphorylation
was detected by 19F solid-state NMR spectroscopy in the
presence and absence of dasatinib. As Figure S6 shows, in the
absence of dasatinib, the Src-416-F2Y sample gave rise to
Figure 4. a) SDS-PAGE of wt Etk (lane 1) and Etk-574-F2Y (lane 2).
b) Western blot experiment using an anti-pTyr antibody showed that
phosphorylated Etk-574-F2Y can be efficiently dephosphorylated by
PTP1B. c) 19F solid-state NMR spectrum of Etk-574-F2Y. Chemical
shifts are referenced to trifluoroacetic acid (TFA) as an internal
standard. d) 19F solid-state NMR spectrum of Etk-574-F2Y-R614A.
a
19F NMR signal at À125.1 ppm, indicating that F2Y at the
416 position was mostly phosphorylated. In the presence of
dasatinib, the 19F NMR signal shifted to À122.4 ppm, indicat-
ing that dasatinib binding to Src triggers a significant
conformation change in its a-loop, resulting in a 2.7 ppm
upfield shift of the 416-pF2Y signal. Our results are consistent
with previous studies which showed that dasatinib binds to the
activated Abl and Src family PTKs,[3j–l] and may provide
valuable insights for designing better PTK inhibitors.[14]
In conclusion, we have demonstrated the highly efficient
genetic incorporation of the UAA F2Y in E. coli, and its use
as a 19F NMR probe for tyrosine phosphorylation. We solved
the structures of F2YRS in the presence and absence of F2Y,
and unraveled the structural basis for the selective recogni-
tion of F2Y by F2YRS. Our new method for probing tyrosine
phosphorylation has numerous advantages versus traditional
strategies. First, tyrosine phosphorylation at a specific site in
intact proteins can be detected and quantified. By contrast,
peptide substrates cannot recapitulate distal interactions
between PTKs and substrate proteins,[12] which are often
essential for activity. Second, samples can be frozen at any
desired time point, followed by 19F NMR spectra acquisition.
This allows for accurate phosphorylation quantification even
in complex protein samples, and quantitative measurement of
the KM and kcat of phosphorylation reactions.[4i] Third, single-
site 19F incorporation and NMR relaxation analysis can be
conveniently applied to illustrate protein dynamic properties,
because of the high sensitivity of 19F chemical shift anisotropy
(CSA) to environment and absence of 19F–19F direct coupling.
Further NMR studies may help to reveal the motions of
eukaryotic and prokaryotic PTK in the active and inactive
states, and dynamic exchange between these states.[13] While
a relatively large amount of protein is needed, our method has
unique advantages over others, since it allows for the study of
dynamic conformation changes in an intact protein, over
a range of time scales. This sensitive, selective, and robust
method for monitoring PTK activation and activity should
crystal structure and mutagenesis studies which showed that
the Arg614Ala mutant has a much decreased overall catalytic
activity,[3g] a molecular model was constructed to suggest that
Arg614 interacts with pTyr574 to unblock the active site,
allowing for peptide and ATP substrate to bind (Figure 3).
However, no direct evidence was provided to support this
model.[3g] To directly test this hypothesis, we expressed the
mutant Etk-574-F2Y-R614A, and again allowed it to auto-
phosphorylate in the same condition as described above. 19F
solid-state NMR spectroscopy revealed that the pF2Y NMR
signal shifted to À122.73 ppm, which was 0.43 ppm downfield
compared to that of Etk-574-F2Y, suggesting that Arg614
does interact directly with pTyr574. Interestingly, the pF2Y/
F2Y signal intensity ratio was 10.8% in Etk-574-F2Y-R614A,
significantly larger than that of Etk-574-F2Y. These results
suggest that while pTyr574 binding to Arg614 render the
active site accessible to substrates, substitution of Arg614 to
Ala does not impair the autophosphorylation activity of Etk
on residue Tyr574. Instead, the Arg614Ala mutation may
cause pTyr574 to bind to an alternative residue Arg572
(Figure S5), occluding the ATP/ADP substrates from binding
to the active site, resulting in a closed and inactive con-
formation which is also less prone to undergo the reverse
pTyr574 dephosphorylation reaction. It was previously shown
that pF2Y undergoes dephosphorylation reaction 1.5 to 2
folds faster than that of pTyr,[6] therefore the pF2Y/F2Y ratios
observed in our experiments are likely underestimates of
pTyr/Tyr ratios. Nevertheless, there is currently no reliable
method available for quantifying pTyr/Tyr ratios, and pF2Y/
F2Y ratios observed in different mutants can provide valuable
mechanistic insights on how each mutation affects the pTyr/
Tyr equilibrium. Interestingly, the 19F signal arising from the
phosphorylated protein is significantly sharper than the 19F
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Received: January 18, 2013
Published online: February 28, 2013
Keywords: NMR spectroscopy · protein–protein interactions ·
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proteins · tyrosine kinase · tyrosine phosphorylation
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