Aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases

Article abstract of DOI:10.1021/ja711125p

Protein tyrosine phosphatases (PTPs) play key roles in the regulation of normal and pathological processes ranging from cell proliferation, differentiation, metabolism, and survival to many human diseases including cancer and diabetes. Functional studies of PTP can be greatly facilitated by small molecule probes that covalently label the active site of a PTP through an activity-dependent chemical reaction. In this article, we characterize phenyl vinyl sulfonate (PVSN) and phenyl vinyl sulfone (PVS) as a new class of mechanism-based PTP probes. PVSN and PVS inactivate a broad range of PTPs in a time- and concentration-dependent fashion. The PVSN- and PVS-mediated PTP inactivation is active site-directed and irreversible, resulting from a Michael addition of the active-site Cys Sγ onto the terminal carbon of the vinyl group. Structural and mechanistic analyses reveal the molecular basis for the preference of PVSN/PVS toward the PTPs, which lies in the ability of PVSN and PVS to engage the conserved structural and catalytic machinery of the PTP active site. In contrast to early α-bromobenzyl phosphonate-based probes, PVSN and PVS are resistant to solvolysis and are cell-permeable and thus hold promise for in vivo applications. Collectively, these properties bode well for the development of aryl vinyl sulfonate/sulfone-based PTP probes to interrogate PTP activity in complex proteomes.

Full text of DOI:10.1021/ja711125p

Published on Web 06/04/2008
Aryl Vinyl Sulfonates and Sulfones as Active Site-Directed
and Mechanism-Based Probes for Protein Tyrosine
Phosphatases
Sijiu Liu, Bo Zhou, Heyi Yang,† Yantao He, Zhong-Xing Jiang, Sanjai Kumar,‡
Li Wu, and Zhong-Yin Zhang*
Department of Biochemistry and Molecular Biology, Indiana UniVersity School of Medicine,
635 Barnhill DriVe, Indianapolis, Indiana 46202
Received December 14, 2007; E-mail: zyzhang@iupui.edu
Abstract: Protein tyrosine phosphatases (PTPs) play key roles in the regulation of normal and pathological
processes ranging from cell proliferation, differentiation, metabolism, and survival to many human diseases
including cancer and diabetes. Functional studies of PTP can be greatly facilitated by small molecule probes
that covalently label the active site of a PTP through an activity-dependent chemical reaction. In this article,
we characterize phenyl vinyl sulfonate (PVSN) and phenyl vinyl sulfone (PVS) as a new class of mechanism-
based PTP probes. PVSN and PVS inactivate a broad range of PTPs in a time- and concentration-dependent
fashion. The PVSN- and PVS-mediated PTP inactivation is active site-directed and irreversible, resulting
from a Michael addition of the active-site Cys Sγ onto the terminal carbon of the vinyl group. Structural
and mechanistic analyses reveal the molecular basis for the preference of PVSN/PVS toward the PTPs,
which lies in the ability of PVSN and PVS to engage the conserved structural and catalytic machinery of
the PTP active site. In contrast to early R-bromobenzyl phosphonate-based probes, PVSN and PVS are
resistant to solvolysis and are cell-permeable and thus hold promise for in vivo applications. Collectively,
these properties bode well for the development of aryl vinyl sulfonate/sulfone-based PTP probes to
interrogate PTP activity in complex proteomes.
sensitivity toward insulin and are resistant to obesity.5,6 In
addition, PTPs can also potentiate, rather than antagonize,
Introduction
Protein phosphorylation, targeted to tyrosine residues, plays
a central role in regulating a diverse range of fundamental
cellular processes, including growth, differentiation, metabolism,
cell cycle progression, cell adhesion and migration, ion channel
activity, immune response, and apoptosis/survival.1 In vivo,
tyrosine phosphorylation is reversible and dynamic and is
governed by the opposing activities of protein tyrosine kinases
and protein tyrosine phosphatases (PTPs). Although the impor-
tance of protein tyrosine kinases in cell physiology has long
been established, the PTPs have garnered increasing recognition
as critical modulators of signaling events regulated by tyrosine
phosphorylation.2 The PTPs constitute a large family of en-
zymes,3 and dysfunction in PTPs has been linked to the etiology
of several human diseases, including cancer, diabetes and
obesity, and autoimmune disorders.4 For example, PTP1B is
implicated as a negative regulator of insulin and leptin-mediated
processes, and mice lacking functional PTP1B exhibit increased
actions of protein tyrosine kinases. Thus, SHP2 is a positive
mediator of growth factor signaling, and gain-of-function
mutations in SHP2 are associated with Noonan syndrome and
several forms of leukemia.7,8
Our current knowledge of the PTPs has been mostly derived
from experiments using gene knockout or overexpression
techniques. As discussed above, plausible biological functions
have been documented for a number of PTPs, and it is clear
that phosphate removal by the PTPs can either enhance or
antagonize a cellular process. Unfortunately, genetic approaches
do not provide an adequate view of the temporal, spatial, and
dynamic properties of the PTPs in cell signaling. Given the large
number and complexity of PTPs in cell physiology, new
strategies are needed for integrated analysis of the PTPs in the
(5) Elchelby, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins, S.;
Lee Loy, A.; Normandin, D.; Cheng, A.; Himms-Hagen, J.; Chan,
C.-C.; Ramachandran, C.; Gresser, M. J.; Tremblay, M. L.; Kennedy,
B. P. Science 1999, 283, 1544–1548.
† Current address: Mount Sinai School of Medicine, 1425 Madison
Avenue, New York, NY 10029.
(6) Klaman, L. D.; Boss, O.; Peroni, O. D.; Kim, J. K.; Martino, J. L.;
Zabolotny, J. M.; Moghal, N.; Lubkin, M.; Kim, Y. B.; Sharpe, A. H.;
Stricker-Krongrad, A.; Shulman, G. I.; Neel, B. G.; Kahn, B. B. Mol.
Cell. Biol. 2000, 20, 5479–5489.
‡ Current address: Queens College, 65-30 Kissena Blvd., Flushing, NY
11367.
(1) Hunter, T. Philos. Trans. R. Soc. London, Ser. B 1998, 353, 583–
605.
(7) Tartaglia, M.; Mehler, E. L.; Goldberg, R.; Zampino, G.; Brunner,
H. G.; Kremer, H.; van der Burgt, I.; Crosby, A. H.; Ion, A.; Jeffery,
S.; Kalidas, K.; Patton, M. A.; Kucherlapati, R. S.; Gelb, B. D. Nat.
Genet. 2001, 29, 465–468.
(2) Tonks, N. K. Nat. ReV. Mol. Cell Biol. 2006, 7, 833–846.
(3) Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Friedberg, I.; Osterman,
A.; Godzik, A.; Hunter, T.; Dixon, J.; Mustelin, T. Cell 2004, 117,
699–711.
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Jung, A.; Hahlen, K.; Hasle, H.; Licht, J. D.; Gelb, B. D. Nat. Genet.
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9
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2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 8251–8260 8251

A R T I C L E S
Liu et al.
sulfonate- and sulfone-based probes for functional characteriza-
tion of the PTPs, thereby enhancing our understanding of the
roles of PTPs in both health and diseases.
Materials and Methods
Materials. Methyl vinyl sulfone (MVS), phenyl vinyl sulfone
(PVS), and phenyl vinyl sulfonate (PVSN) were obtained from
Aldrich. PEG and buffers for crystallization were purchased from
Hampton Research Co. p-Nitrophenyl phosphate (pNPP) was
purchased from Fluke Co. Antiphosphotyrosine (pY20) polyclonal
antibody was from B. D. Biosciences. Dithiothreitol (DTT),
iodoacetamide, urea, ammonium bicarbonate, and acetonitrile were
provided by Fisher. Formic acid was purchased from E. Merck.
R-Cyano-4-hydroxycinnamic acid and modified sequencing grade
trypsin were purchased from Sigma. Ziptipc18 was obtained from
Millipore. Dulbecco’s modified Eagle’s medium (DMEM) and fetal
bovine serum were purchased from ATCC. Penicillin and strepto-
mycin were purchased from Cellgrow. EDTA-free complete pro-
tease inhibitor cocktail tablets were purchased from Roche. All other
chemicals and reagents were of the highest grade commercially
available.
Figure 1. Structures of methyl vinyl sulfone, phenyl vinyl sulfone, phenyl
vinyl sulfonate, and benzyl vinyl sulfonate.
whole proteome. To interrogate the functional complexity of
the proteomes, powerful chemical reagents have been developed
for targeted analysis of individual protein families.9 In particular,
mechanism-based probes are available for covalent labeling of
the cysteine and serine hydrolases, providing new insights into
our understanding of these two families of proteases in cell
biology and in diseases.10,11 In addition to profiling the
functional state of enzymes, mechanism-based probes are also
seeing application in drug discovery, target identification, and
discovery of previously uncharacterized enzyme activity.9
To develop class-selective PTP probes, 4-fluromethylaryl
phosphate was initially examined12 as its hydrolysis generates
a highly reactive quinone methide intermediate, which alkylate
nucleophiles at, or near, the phosphatase active site.13 Unfor-
tunately, 4-fluromethylaryl phosphate is not specific for the
PTPs, as the diffusible, unmasked quinone methide electrophile
will nonspecifically label any nearby proteins. Consequently,
chemical probes based on 4-fluromethylaryl phosphate lack the
specificity required for profiling PTP activity. More recently,
we reported several R-bromobenzyl phosphonate-based probes
with extremely high selectivity for the PTPs.14,15 When attached
to appropriate affinity or fluorescent tags, these probes enable
the identification and visualization of the labeled PTPs in
complex proteomes. One major drawback for R-bromobenzyl
phosphonate is the exceedingly low membrane permeability,
which limits its usefulness as a research tool to globally
characterize PTP activity in live cells.
The current dearth of cell-permeable covalent probes for PTPs
stands as a significant challenge for investigators interested in
mechanism-based profiling of this large enzyme class. As a first
step toward the development of small molecule modulators of
PTP activity inside the cell, we describe the characterization of
aryl vinyl sulfonates and sulfones (Figure 1) as cell-permeable,
mechanism-based probes for the PTPs. We show that the aryl
vinyl sulfonates and sulfones inactivate a broad range of PTPs
in a time- and concentration-dependent fashion. We establish
that PTP inactivation by these compounds is active site-directed
and irreversible. We provide evidence that these probes form a
covalent adduct with PTPs, involving the active site Cys residue.
These properties bode well for the application of the aryl vinyl
Synthesis of Benzyl Vinyl Sulfonate. 2-Chloro-1-ethane-sul-
fonylchloride (0.2 mL, 1.91 mmol) dissolved in anhydrous dichlo-
romethane (5 mL) and benzyl alcohol (0.2 mL, 1.93 mmol) was
treated, at 0 °C, with triethylamine (0.54 mL, 3.87 mmol). After
0.5 h, the reaction mixture was concentrated and the crude residue
was purified by flash chromatography on silica gel (6% ethyl acetate
in hexanes) to provide benzyl vinyl sulfonate (313 mg, 83% yield)
as colorless oil. 1H NMR (300 MHz, CDCl3) δ 7.382 (m, 5H),
6.516-6.362 (m, 2H), 6.066 (d, J ) 8.7 Hz, 1H), 5.149 (s, 2H);
LC-ESI MS (m/z): 199 (M + 1).
16
Synthesis of Alkyne-Labeled Biotin. N-(3-Dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride (920 mg, 4.8 mmol)
was added to a solution of biotin (1 g, 4.1 mmol) and N-
hydroxysuccinimide (510 mg, 4.4 mmol) in DMF (50 mL). The
reaction solution was concentrated after 24 h at room temperature.
The residue was washed by methanol three times, and the solvent
was removed to yield a white solid, which was directly dissolved
in DMF (100 mL). To this solution was added 0.48 mL of
propargylamine (7.0 mmol) and 1.4 mL of triethylamine (10 mmol),
and the solution was stirred at room temperature for 24 h.
Subsequently, the reaction solution was concentrated in vacuum.
Chromatography of the residue (MeOH/CHCl3, 10/1) gave the
alkyne-labeled biotin (682 mg, yield 61%). 1H (CD3OD, 500 MHz)
δ 1.76 (quintet, J ) 7.55 Hz, 2H), 1.90-2.10 (m, 4H), 2.53 (t, J
) 7.3 Hz, 2H), 2.89 (t, J ) 2.5 Hz, 1H), 2.93-3.03 (m, 4H), 3.24
(dd, J ) 12.7 Hz, 5 Hz, 1H), 3.52 (m, 1H), 4.26 (d, J ) 2.6 Hz,
2H), 4.62 (dd, J ) 7.8 Hz, 4.5 Hz, 1H), 4.80 (dd, J ) 7.9 Hz, 4.9
Hz, 1H); LC-MS (ESI) m/z: 282.1 [M + 1]+.
Synthesis of Azide-Tagged PVSN Probe. To a stirred solution
of 4-(bromomethyl)phenol (18.7 g, 100 mmol) in DMF (200 mL)
was added sodium azide (7.8 g, 120 mmol), and the resulting
solution was stirred for 4 h at room temperature. Then, the reaction
mixture was diluted with EtOAc (600 mL) and washed with brine
(100 mL, three times). The organic layer was dried with Na2SO4,
filtrated through a pad of silica gel, and concentrated to give crude
4-(azidomethyl)phenol as a clear oil (11.3 g, yield 76%), which
was used without further purification. 2-Chloroethanesulfonylchlo-
ride (15.0 g, 92 mmol) was added to a stirred solution of
4-(azidomethyl)phenol (11.3 g, 76 mmol) in CH2Cl2 (400 mL) at
0 °C. Then, ice-cold trimethylamine (39.9 mL, 276 mmol) was
added in one portion at 0 °C. After 0.5 h, the reaction mixture was
washed with cold 10% aqueous Na2CO3 (100 mL, three times),
2N HCl (100 mL), and water (100 mL), and the organic layer was
dried with Na2SO4 and concentrated to give a residue that was
purified by flash chromatography on silica gel (CH2Cl2/hexane )
(9) Evans, M. J.; Cravatt, B. F. Chem. ReV. 2006, 106, 3279–3301.
(10) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 14694–14699.
(11) Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M.
Chem. Biol. 2000, 7, 569–581.
(12) Lo, L.-C.; Pang, T.-L.; Kuo, C.-H.; Chiang, Y.-L.; Wang, H.-Y.; Lin,
J.-J. J. Proteome Res. 2002, 1, 35–40.
(13) Myers, J. K.; Widlanski, T. S. Science 1993, 262, 1451–1453.
(14) Kumar, S.; Zhou, B.; Liang, F.; Wang, W.-Q.; Huang, Z.; Zhang, Z.-
Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7943–7948.
(15) Kumar, S.; Zhou, B.; Liang, F.; Yang, H.; Wang, W.-Q.; Zhang, Z.-
Y. J. Proteome Res. 2006, 5, 1898–1905.
(16) Lin, P.-J.; Ueng, S.-H.; Yu, S.-C.; Jan, M.-D.; Adak, A. K.; Yu, C.-
C.; Lin, C.-C. Org. Lett. 2007, 9, 2131–2134.
9
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Aryl Vinyl Sulfonate/Sulfone-Based PTP Probes
A R T I C L E S
Table 1. Crystallographic Data and Refinement Statistics
1/3) to give 4-(azidomethyl)phenyl ethenesulfonate (azide-tagged
1
PVSN probe) (14.75 g, yield 67%). H NMR (500 MHz, CDCl3)
YopH·PVS
YopH·PVSN
δ 7.36 (m, 2H), 7.26 (m, 2H), 6.68 (dd, J ) 16.7 Hz, 9.9 Hz, 1H),
6.38 (d, J ) 16.7 Hz, 1H), 6.19 (d, J ) 9.9 Hz, 1H), 4.38 (s, 2H);
13C NMR (125.9 MHz, CDCl3) δ 149.2, 134.8, 132.1, 131.8, 129.5,
122.6, 53.9; MS (ESI) m/z: 212 ((M + 1 - N2)+), 197 ((M + 1 -
N3)+).
PTPs and Non-PTP Proteins. The catalytic domain (residues
163-468) of YopH was expressed in Escherichia coli and purified
as described.17 Other recombinant PTPs were expressed and purified
as described previously.18–20 Non-PTP enzymes were purchased
from Sigma-Aldrich and stored at -20 °C.
Kinetic Characterization of PTP Inactivation by Vinyl Sulf-
onates and Sulfones. PTP inactivation by the vinyl sulfonates and
sulfones was studied at 25 °C in a pH 6 buffer containing 50 mM
sodium succinate, 1 mM EDTA, and ionic strength of 150 mM
adjusted with NaCl. At appropriate time intervals, aliquots of 2 µL
were removed from the reaction and added into a 200-µL solution
containing 20 mM pNPP in pH 6.0 buffer at 30 °C.14 The kinetic
parameters of the inactivation reaction were obtained by fitting the
data to the following equations:
Crystal Parameters
space group
P212121
P212121
Cell Dimensions (Å)
a
b
c
46.8
54.0
96.9
49.2
55.6
97.5
Data Collection
resolution (Å)
50-2.0
17150
94.6
3.2
0.097
50-2.0
16289
90.3%
3.5
no. of unique reflections
completeness (%)
redundancy
a
Rmerge
0.047
Refinement
resolution limit (Å)
no. of reflections used (F g 2δ (F))
no. of protein atoms
no. of inhibitors
50-2.0
14611
2166
1
164
50-2.2
12279
2166
1
94
18.0/22.6
no. of waters
c
Rworkb/Rfree
20.5/25.3
rms Deviations from Ideal Geometry
bond length (Å)
At
A∞
A0
A0 - A∞
)
-
exp(-kobs · t)
(1)
(2)
0.0053
1.28
27.2
0.0060
1.24
23.6
(
)
A0
A0
bond angle (deg)
average B-factors (Å2)
ki × [I]
kobs
)
a Rmerge ) ΣhΣi|I(h)i - I(h) |/ΣhΣiI(h)i Rwork ) Σh|F(h)calcd - F(h)obsd|/
ΣhF(h)obsd, where F(h)calcd and F(h)obsd were the refined calculated and
observed structure factors, respectively. Rfree was calculated for
randomly selected 3.3% (YopH ·PVS) and 2.6% (YopH ·PVSN) of the
reflections that were omitted from refinement.
b
KI + [I]
LC-MS Analysis. An electrospray ion trap mass spectrometer
(LCQ DECA XP plus, Thermo Finnigan) coupled with an online
HPLC (LC10 AD VP, Shimadzu) was used for measurement of
the molecular weight of proteins. A 1.0 × 25 mm C8 column (5-
µm particle diameter, 300 Å pore size, VYDAC) with mobile phases
A (0.1% formic acid in water) and B (0.1% formic acid in
acetonitrile) was used to desalt and separate proteins by a linear
gradient 5-45% of mobile phase B over 30 min at a flow rate of
25 µL/min. Desalted proteins were eluted directly to mass spec-
trometer for molecular weight measurement. Molecular weights of
proteins were calculated by deconvolution of the multicharged ions
formed by electrospray ionization (ESI) using Bioworks software
(Thermo Finnigan).
c
a
of 15-45% B in 30 min. A LCQ Deca XP ion-trap mass
spectrometer (Thermo Finnigan) was used to analyze peptides eluted
from the column. The instrument acquired one full-scan mass
spectrum (300-2000 m/z) followed by three data-dependent MS/
MS spectra at 36% normalized collision energy. Dynamic exclusion
was configured to minimize the number of replicate MS/MS spectra
by excluding the m/z of the previous 25 precursors selected for
collision-induced dissociation.
Preparation and Crystallization of the YopH·PVSN and
YopH·PVS Complexes. YopH (10 mL, 1 mg/mL in 20 mM Bis-
Tris, pH 6.0 buffer) was mixed with 30 µL of PVSN or PVS stock
solution (100 mM, dissolved in DMSO) at 4 °C for covalent
labeling. Mass spectrometry was used to track covalent adduct
formation. After 7 days of reaction with PVSN or PVS at 4 °C,
YopH was found to be completely modified by the compounds.
The YopH solution was then concentrated at ∼7 mg/mL, frozen
by liquid nitrogen, and stored at -80 °C for crystallization. Crystals
of YopH ·PVSN (coordinates deposited in the Protein Data Bank,
accession number 3BLT) and YopH ·PVS (PDB accession number
3BLU) were obtained at 18 °C by vapor diffusion in hanging drops.
The conditions for getting YopH ·PVSN and YopH ·PVS crystals
were similar. The protein drops were equilibrated against a reservoir
solution containing 25% w/v poly(ethylene glycol) 3350, 100 mM
NaCl, and 100 mM HEPES buffer (pH 7.0-8.0). The drops
consisted of protein at a concentration of 7 mg/mL, diluted by an
equal volume of the reservoir solution. The crystals of YopH ·PVSN
and YopH ·PVS belong to space group P212121 (Table 1).
X-ray Data Collection and Structural Determination. For
X-ray data collection, the crystals were flash-cooled by liquid
nitrogen in their original PEG solutions used for crystallization.
X-ray data of YopH·PVSN were collected at 100 K on a Rigaku
RU-300 rotating anode generator (Rigaku/MSC) equipped with
focusing mirrors (MSC/Yale) and an R-AXIS IV++ image plate
detector; X-ray data of YopH ·PVS were collected at 100 K on the
F2 station beamline of MacCHESS. The data were processed using
the program HKL.21 Statistics of the data processing are provided
in Table 1.
Protein Digestion and MALDI-TOF Analysis. YopH proteins
were incubated at 56 °C for 30 min in a solution containing 10
mM DTT and 8 M urea. Subsequently, 50 mM iodoacetamide was
introduced to alkylate the denatured and reduced proteins. The
protein was digested with trypsin (protein/enzyme, 50/1) at 37 °C
overnight. The resulting peptides were desalted using Ziptipc18
before MALDI-TOF experiments. A volume of 0.5 µL of the
desalted peptides was mixed with 0.5 µL of saturated R-cyano-4-
hydroxycinnamic acid solution in 50% aqueous acetonitrile contain-
ing 0.1% trifluoroacetic acid and spotted on a MALDI-TOF target
plate. The sample matrix solution was allowed to air dry at room
temperature. Voyager DE STR MALDI-TOF (Applied Biosystems)
was used to obtain mass spectra in positive and reflector mode.
LC-MS/MS Analysis. A 180-µm id × 15 cm column packed
with 3-µm 100 Å C18 stationary phase was used for chromatog-
raphy separation in an Ultimate HPLC system (Dionex). The pump
flow rate was split 1:125 for a column flow rate of 1 µL/min. The
column effluent was directly electrosprayed using the orthogonal
metal needle source without further splitting. Mobile phase A was
0.1% formic acid in water, and phase B was 0.1% formic acid in
acetonitrile. The separation of peptides was achieved with a gradient
(17) Zhang, Z.-Y.; Clemens, J. C.; Schubert, H. L.; Stuckey, J. A.; Fischer,
M. W. F.; Hume, D. M.; Saper, M. A.; Dixon, J. E. J. Biol. Chem.
1992, 267, 23759–23766.
(18) Shen, K.; Keng, Y.-F.; Wu, L.; Guo, X.-L.; Lawrence, D. S.; Zhang,
Z.-Y. J. Biol. Chem. 2001, 276, 47311–47319.
(19) Zhou, B.; Zhang, Z.-Y. J. Biol. Chem. 2002, 277, 13889–13899.
(20) Sun, J.-P.; Wu, L.; Fedorov, A. A.; Almo, S. C.; Zhang, Z.-Y. J. Biol.
Chem. 2003, 278, 33392–33399.
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A R T I C L E S
Liu et al.
To determine the structure of YopH ·PVSN, YopH ·p-nitrocat-
echol sulfate (pNCS) (PDB entry code 1PA9),20 omitting the pNCS
and solvent molecules, was used as a search model to calculate a
difference Fourier map for YopH ·PVSN with the coefficients 2Fo
- Fc and calculated phases (where Fo and Fc are the observed and
calculated structure factors, respectively). The structures were
refined to 2.2 Å resolution with the program CNS,22 first using
simulated annealing at 3000 K, and then alternating positional and
individual temperature factor refinement cycles. The progress of
the refinement was evaluated by the improvement in the quality of
the electron density maps and the reduced values of the conventional
R factor (R ) Σh|Fo| - |Fc|/Σh|Fo|) and the free R factor (2.6% of
the reflections omitted from the refinement).23 The electron density
maps were inspected, and the model was modified on an interactive
graphics workstation with the program O.24 Finally, water molecules
were added gradually as the refinement progressed. They were
assigned in the |Fo| - |Fc| difference Fourier maps with a 3δ cutoff
level for inclusion in the model. The structure of YopH ·PVS was
solved using the structure of YopH ·PVSN without the solvent
molecules and PVSN as the search model. The structure was refined
to 2.0 Å resolution with the program CNS.22 The refinement
statistics are shown in Table 1.
using anti-Biotin antibody and anti-His6 antibody. The second gel
was Coomassie blue stained for protein visualization.
Results and Discussion
Unlike protein kinases, where tyrosine-specific and serine/
threonine-specific kinases share sequence identity, the PTPs
show no structural similarity with serine/threonine phosphatases
or the broad specificity acid or alkaline phosphatases. The
hallmark that defines the PTP superfamily is the active-site
sequence (H/V)C(X)5R(S/T), also called the PTP signature
motif, in the catalytic domain. The PTPs share a conserved
active site that recognizes phosphotyrosine (pTyr) and a common
catalytic mechanism that utilizes a highly reactive nucleophilic
Cys residue.26 This Cys (Cys403 in the Yersinia PTP YopH)
displays an unusually low pKa of ∼527 and is situated at the
bottom of the pTyr-binding pocket (i.e., the active site) such
that its Sγ atom is poised 3 Å from the phosphorus atom of
pTyr.28 In the catalytic mechanism, the active-site Cys initiates
a nucleophilic attack on the phosphorus atom, leading to the
formation of a thiophosphoryl enzyme intermediate. This step
is assisted by a general acid (Asp356 in YopH), which is in
close proximity to the scissile phenolic oxygen of pTyr. The
active-site arginine (Arg409 in YopH) participates in binding
of the pTyr substrate and stabilizes the transition state during
catalysis. Hydrolysis of this covalent enzyme intermediate then
completes the catalytic cycle.26
The involvement of Cys for nucleophilic catalysis has
important implications for the development of mechanism-based
PTP probes, where design strategies may exploit the extraor-
dinary reactivity of the active-site thiol group. In this regard,
we note that a number of vinyl sulfone derivatives act as
irreversible inhibitors of the cysteine proteases.29–31 Given the
intrinsic affinity of the PTP active-site pocket for pTyr, we
reasoned that aryl vinyl sulfones or sulfonates may serve as
pTyr surrogates and function as mechanism-based PTP inacti-
vators. In the following, we describe a structural and mechanistic
study of PVSN and PVS as mechanism-based, cell-permeable
PTP probes.
PVSN and PVS Are Active Site-Directed and Irreversible
PTP Inactivators. To determine whether PVSN and PVS can
function as mechanism-based PTP probes, we first examined
their effect on PTP activity using pNPP as a substrate. Both
compounds inactivated the Yersinia PTP, YopH, in a time- and
concentration-dependent first-order process (Figure 2A). Similar
results were obtained with several PTPs, including PTP1B,
HePTP, DEP-1, VHR, PRL1, and the low molecular weight
PTP (data not shown). Inactivation of the PTPs by PVSN and
PVS appeared irreversible as extensive dialysis and/or buffer
exchange of the reaction mixture failed to recover enzyme
activity. Analysis of the pseudo-first-order rate constant as a
function of inhibitor concentration showed that PVSN-mediated
YopH inactivation displayed saturation kinetics (Figure 2B),
yielding values for the equilibrium binding constant KI and the
Effect of PVSN and PVS on Cellular Phosphotyrosine Level.
COS-7 cells were grown in DMEM supplemented with 10% fetal
bovine serum, penicillin (50 units/mL), and streptomycin (50 µg/
mL) under a humidified atmosphere containing 5% CO2. Nearly
80% confluent COS-7 cells were treated for 1 h with 0.5 and 1
mM PVSN, PVS, MVS, R-bromobenzyl phosphonate (BBP), or 1
mM sodium vanadate. Cells were washed twice using ice-cold PBS
and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 1%
Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM
Na3VO4, 15 mM NaF, and an EDTA-free complete protease
inhibitor cocktail. Cell lysates were precleared by centrifugation
at 15000 rpm for 15 min. Protein concentration was determined
by the Bradford method. Protein samples (50 µg) were resolved
by 10% SDS-PAGE and subjected to immunoblotting with an-
tiphosphotyrosine antibody 4G10.
PTP Labeling with the PVSN Probe by Click Chemistry.
Specific labeling of the desired PTP in a complex proteome with
the azide-tagged PVSN probe and click chemistry was carried out
using a procedure similar to that described previously.25 Cell lysates
from E. coli transformed with or without His6-HePTP (50 µg of
total protein) were incubated with 20 µM azide-tagged PVSN probe
for 30 min at room temperature in a pH 6.0 buffer (50 mM sodium
succinate, 1 mM EDTA, adjusted to the ionic strength of 150 mM
with NaCl). Each protein sample (85 µL) was then treated with
0.125 mM alkyne-labeled biotin (2.5 µL, 5 mM stock in DMSO),
followed by 1.25 mM tris(2-carboxyethyl)phosphine (TCEP) (2.5
µL, 50 mM stock in water), 0.127 mM tris(benzyltriazolylmethy-
l)amine (TBTA) (7.5 µL, 1.7 mM in DMSO), and 1.25 mM CuSO4
(2.5 µL, 50 mM stock in water). The click reaction was allowed to
proceed at room temperature on a shaker for 1 h. The reaction was
then quenched by the addition of one volume of 2 × SDS loading
buffer and heated at 90 °C for 5 min. Each sample was divided
into halves, which were separated on a 12% SDS-PAGE (10 µg of
total protein per lane). The samples from one gel were transferred
to a nitrocellulose membrane and subjected to Western blot analysis
(26) Zhang, Z.-Y. Prog. Nucleic Acid Res. Mol. Biol. 2003, 73, 171–220.
(27) Zhang, Z.-Y.; Dixon, J. E. Biochemistry 1993, 32, 9340–9345.
(28) Jia, Z.; Barford, D.; Flint, A. J.; Tonks, N. K. Science 1995, 268,
1754–1758.
(21) Otwinowski, Z.; Minow, W. Methods Enzymol. 1997, 176, 307–326.
(22) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Acta Crystallogr., Sect. D 1998, 54, 905–921.
(29) Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D. J. Med. Chem.
1995, 38, 3193–3196.
(30) Roush, W. R.; Gwaltney, S. L., II; Cheng, J.; Scheidt, K. A.;
McKerrow, J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994–
10995.
(23) Bru¨nger, A. T. Nature 1992, 355, 472–474.
(24) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, G. J. Acta
Crystallogr., Sect. A 1991, 47, 110–119.
(25) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535–546.
(31) Frankel, B. A.; Bentley, M.; Kruger, R. G.; McCafferty, D. G. J. Am.
Chem. Soc. 2004, 126, 3404–3405.
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modifying agents, denatured in urea, reduced with DTT,
alkylated with iodoacetamide, and digested with trypsin. The
resulting peptides were analyzed by MALDI-TOF mass spec-
trometry in reflector mode. Peptide mass fingerprints were
shown in Figure 3B. Peaks 1050.6, 1161.5, and 1177.2 were
assigned to peptide 397LRPVIHCR404, in which the active-site
Cys403 was covalently modified by iodoacetamide, PVS, or
PVSN. To further confirm that Cys403 was the site of PVS or
PVSN attachment, peptides from trypsin-digested YopH labeled
with or without PVS or PVSN were analyzed by tandem mass
spectrometry. Figure 3C shows the MS/MS spectra of the
iodeoacetamide-, PVS-, and PVSN-modified 397LRPVIHCR404
peptides (peaks 1050.6, 1161.5, and 1177.2 in Figure 3B). The
mass differences between fragment ions b6 and b7 unequivocally
identified Cys403 as the site of covalent modification. The
results from MALDI-TOF and electrospray MS/MS experiments
are consistent with the formation of a covalent adduct between
YopH and PVS/PVSN involving the active-site Cys.
Structural Basis of YopH Inactivation by PVSN and PVS.
To gain further insights into the mechanism of YopH inactiva-
tion by PVSN and PVS, we determined the crystal structures
of YopH ·PVSN and YopH ·PVS complexes. Crystals of the
YopH · inactivator complexes were obtained at 18 °C by the
hanging-drop vapor diffusion method with 7 mg/mL of YopH
that was fully modified by either PVSN or PVS (for details,
see Materials and Methods). Diffraction data were then col-
lected, and the YopH ·PVSN and YopH ·PVS structures were
solved by molecular replacement using coordinates of YopH ·p-
nitrocatechol sulfate complex21 as a search model and refined
to 2.2 and 2.0 Å, respectively (Table 1). The final models for
the YopH·PVSN and YopH ·PVS complexes included YopH
residues 186-468 and all atoms in PVSN and PVS. The overall
structure of YopH ·PVSN is comparable to the previously
determined ligand-free YopH structure,32 with root mean square
derivation (rmsd) between all R-carbon positions of the two
structures being 0.621 Å excluding the flexible WPD loop
(residues 351-359). The major difference between these two
structures is electron density in the PTP active site corresponding
to PVSN, which is covalently attached to YopH via a thioether
linkage between Cys403 Sγ and the terminal carbon of the vinyl
group (Figure 4). The presence of electron density for this
covalent adduct between PVSN and YopH was confirmed by
analyzing the 2Fo - Fc and Fo - Fc difference Fourier maps
with contour levels of 1.0 and 2.5 δ, respectively (Figure 4B).
Similar observations were also made with the YopH·PVS
complex. The bond lengths for the thioether linkages between
YopH/Cys403 and PVSN and PVS are 1.73 and 1.82 Å,
respectively, which are in agreement with literature values for
the C-S bond. Collectively, the structural data further support
the conclusion that PVSN and PVS specifically inactivate the
PTPs by modifying the active-site Cys residue.
Figure 2. Kinetics of YopH inactivation by phenyl vinyl sulfonate. (A)
Time and concentration dependence of YopH inactivation by phenyl vinyl
sulfonate. The experimental points are represented by various symbols, and
the line connecting them is the fitted line to eq 1 to yield pseudo-first-order
rate constant kobs. Probe concentrations were as follows: (, 1 mM; ∆, 2
mM; 0, 3 mM; 9, 5 mM; ], 7.5 mM; and 2, 10 mM. (B) Concentration
dependence of the pseudo-first-order rate constant kobs for phenyl vinyl
sulfonate-mediated YopH inactivation. The points are experimental, and
the line connecting them is the fitted line to eq 2.
inactivation rate constant ki of 0.29 ( 0.06 mM and 0.061 (
0.002 min-1, respectively. Similar saturation kinetics was also
observed for YopH inactivation by PVS (0.35 ( 0.06 mM and
0.080 ( 0.004 min-1 for KI and ki). Thus, PVSN and PVS were
equally effective at inhibiting PTP activity. These results suggest
that PVSN and PVS are active site-directed affinity agents whose
mode of action likely involves at least two steps: binding to
the PTP active site followed by covalent modification of active-
site residue(s). Further evidence in support of the inactivation
being directed to the active site included the observation that
arsenate, a competitive PTP inhibitor, was able to protect YopH
from the PVSN-mediated inactivation (Figure S1).
PVSN and PVS React with the Active-Site Cys and Form
a Covalent Adduct with the PTPs. To directly demonstrate that
PVSN and PVS inactivate the PTPs by covalent modification,
we analyzed recombinant YopH treated with either PVSN or
PVS using mass spectrometry. The molecular weight for the
catalytic domain of YopH (residues 163-468) measured by an
electrospray ion trap mass spectrometer was 33 514, which is
in close agreement with the theoretical value (33 513). The
measured molecular weights of 33 680 and 33 700 for the PVS-
and PVSN-modified YopHs indicated a mass shift of 166 and
186, which correspond closely to the expected mass shift
resulting from covalent attachment of PVS (mass 168.2) and
PVSN (mass 184.2), respectively (Figure 3A). To identify the
site of modification, YopH was incubated with or without the
In addition to revealing the nature of covalent linkage between
YopH and PVSN/PVS, the structures also provide high-
resolution views of noncovalent interactions between the active
site of YopH and PVSN/PVS. As shown in Figure 5A, PVSN
occupies the active-site pocket of YopH in a manner that is
very similar to the way the pTyr mimetic p-nitrocatechol sulfate
binds to YopH.21 PVSN is inserted into an ∼10 Å deep crevice
that is capped by a loop from the end of strand ꢀ8 and to the
first turn of helix R5, which has been termed the phosphate-
(32) Stuckey, J. A.; Schubert, H. L.; Fauman, E.; Zhang, Z.-Y.; Dixon,
J. E.; Saper, M. A. Nature 1994, 370, 571–575.
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A R T I C L E S
Liu et al.
Figure 3. Mass spectrometry analyses of YopH modification by PVSN and PVS. (A) Deconvoluted ESI-ion trap mass spectra of YopH (a), YopH labeled
with PVS (b) or PVSN (c). (B) MALDI-TOF spectra of trypsin-digested peptides from YopH modified by PVSN (a), PVS (b), and unmodified YopH (c).
Peaks 1050.6, 1161.5, and 1177.2 were assigned to peptide 397LRPVIHCR404, in which the active-site Cys403 was covalently modified by iodoacetamide,
PVS, or PVSN. (C) MS/MS spectra of 397LRPVIHCR404 from the trypsin-digested YopH treated with iodoacetamide (a), PVS (b), or PVSN (c). The mass
differences between fragment ions b6 and b7 indicate that Cys403 was the site of covalent attachment by iodoacetamide, PVS, or PVSN in YopH.
binding loop or P-loop (residues 404-409). The sulfonyl moiety
is surrounded by main-chain amides of the P-loop, which donate
four hydrogen bonds, the side chain of Arg409, which forms
two salt bridges with the O2 atom, and a structural water
molecule (Wat1) that is hydrogen-bonded to oxygen O2.
Stabilized by polar interactions with the side chains of the
invariant Asp356, Gln446, and Gln450, and the main-chain
amide from Gln357, Wat1 is conserved and has been observed
in a number of PTP inhibitor/substrate complexes.21,28,32 At the
center of the P-loop lies the catalytic Cys403, which forms a
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Figure 4. Crystal structure of YopH in complex with PVSN. (A) Overall
structure of the YopH ·PVSN complex. R-Helixes are shown in cyan, and
ꢀ-sheets are shown in yellow. The P-loop and WPD-loop are highlighted
in red and orange, respectively. Cys403 and PVSN are shown in stick model,
and the covalent C-S bond between PVSN and Cys403 is depicted in blue.
(B) Electron density map for the YopH ·PVSN complex. The electron
density map around PVSN and Cys403 was contoured at the 1.0 δ level in
blue. Atoms were colored according to atom type (carbon in yellow, oxygen
in red, sulfur in orange). P-loop was also shown with transparent cartoon
in green color.
covalent bond with the vinyl group in PVSN. As observed in
the YopH·p-nitrocatechol sulfate complex, the highly flexible
WPD-loop adopts a closed conformation in the YopH ·PVSN
structure. In this closed conformation, the side chain of the
general acid Asp356 makes two hydrogen bonds with the O2
sulfonyl oxygen and the structural water. In addition to the polar
interactions, there are also van der Waals contacts between
YopH and the inhibitor. The phenyl ring of PVSN makes
extensive hydrophobic contacts with side chains of residues
lining up the active-site cavity (i.e., pTyr binding pocket),
including Phe229, Ile232, Arg404, Ala405, and Val407.
The overall structure of YopH ·PVS is very similar to that of
YopH·PVSN (Figure 5B), with rmsd between all R-carbon
positions of the two structures at 0.365 Å. Because of the extra
phenolic oxygen, the sulfonyl group in PVSN moves 0.7 Å
deeper into the YopH active site and rotates 15° to the right
along the phenyl ring-sulfonyl moiety axis (Figure 5C). As a
consequence, the H-bonding pattern between the PVSN sulfonyl
group and the YopH active site is different from that observed
in the YopH ·PVS structure, but the total number of hydrogen
bonds in both complexes remains the same. For example, O1
in PVSN forms three hydrogen bonds with the main chain
amides of Val407, Gly408, and Arg409, whereas O1 from PVS
makes three hydrogen bonds with the main-chain NH groups
of Gly406, Val407, and Gly408. Similarly, O2 from PVSN
forms a hydrogen bond with the main-chain amide of Arg409,
Figure 5. Detailed interactions between YopH and PVSN/PVS. (A)
Stereoview of the interactions between PVSN and YopH. Hydrogen bonds
and polar interactions are highlighted with dash lines in red. The covalent
bond between PVSN and Cys403 is shown in orange. (B) Stereoview of
the interactions between PVS and YopH. Hydrogen bonds and polar
interactions are highlighted with dash lines in red. The covalent bond
between PVS and Cys403 is shown in orange. (C) Superposition of the
YopH·PVSN and YopH ·PVS complexes. Only residues in YopH ·PVSN
are shown with stick model in atom color: carbon, gray; oxygen, red; sulfur,
orange. The P-loop is also shown in cyan cartoon. Carbon atoms of PVSN
and PVS are shown in yellow and green, respectively. Superposition was
calculated with active-site residues and WPD-loop but did not include the
ligands.
another one with the side chain of Asp356, and two salt bridges
with the side chain of Arg409, whereas O2 from PVS forms
two hydrogen bonds with the main-chain amides of Gly408 and
Arg409, an additional hydrogen bond with the side chain of
Asp356, and one salt bridge with the side chain of Arg409
(Figure 5A,B). Interestingly, the phenyl ring of PVS occupies
almost the same position and is engaged in nearly identical van
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A R T I C L E S
Liu et al.
Figure 6. Mechanism of YopH inactivation by PVSN.
Table 2. Summary of the Pseudo-First-Order Rate Constants
der Waals interactions with residues Phe229, Ile232, Arg404,
Ala405, and Val407 as observed in the YopH ·PVSN structure.
ThesestructuralsimilaritiesbetweenYopH ·PVSNandYopH ·PVS
are consistent with the nearly identical kinetics of YopH
inactivation exhibited by PVSN and PVS.
(kobs) for PTP Inactivation by 5 mM PVSN
PTPs
kobs (min-1
)
YopH
0.055 ( 0.004
0.047 ( 0.004
1.66 ( 0.14
PTP1B
HePTP
DEP1
PRL1
VHR
Mechanism and Specificity of PTP Inactivation by PVSN
and PVS. It is clear from the kinetic, mass spectrometry, and
X-ray crystallographic studies that PVSN and PVS can serve
as efficient pTyr surrogates and specifically modify the YopH
active-site Cys403. Together, our data indicate that PVSN and
PVS inactivate the PTPs by electrophilic capture of the active-
site Cys via 1,4-conjugate addition, forming a stable thioether
adduct with the enzyme (Figure 6). A number of conserved
structural features within the PTP active site contribute to
specific PTP inactivation by aryl vinyl sulfonates/sulfones. The
hydrophobic interactions between the phenyl moiety and the
pTyr pocket are clearly important for the sulfone-mediated PTP
inactivation because MVS (Figure 1) had no appreciable effect
on the YopH-catalyzed pNPP reaction even after prolonged
incubation (80 min) at 20 mM inhibitor concentration. We also
noted that the first-order rate of inactivation by benzyl vinyl
sulfonate (Figure 1) was 4.6-fold lower than that of PVSN. Since
the intrinsic chemical reactivity of the vinyl sulfonate group is
expected to be the same within the closely related benzyl and
phenyl vinyl sulfonates, we attribute this decrease in rate to
less favorable hydrophobic interactions between the benzyl
group and the YopH active site because of the presence of an
additional methylene group.
In addition to hydrophobic interactions between the phenyl
ring and the pTyr binding pocket, the reactivity of PVSN and
PVS toward YopH is further enhanced by specific polar
interactions between the sulfonyl oxygens and main-chain
amides from the P-loop and side chains of the invariant catalytic
residues Asp356 and Arg409. In particular, Asp356 functions
as the general acid in the PTP reaction by donating a proton to
the phenolic oxygen in the leaving group.26 In the YopH ·PVS
and YopH·PVSN structures, the side chain of Asp356 makes a
hydrogen bond with the O2 oxygen in PVS/PVSN (Figure 5).
In addition, Asp356 also contributes to the stabilization of Wat1,
which is in hydrogen-bonding distance to O2. We envisaged
that removal or weakening of these polar interactions would
reduce the reactivity of PVSN/PVS toward YopH. We have
determined the crystal structure of YopH/D356A bound to
PVSN, which revealed that PVSN is covalently attached to
Cys403 but Wat1 is absent in this structure (coordinates
deposited in the Protein Data Bank, accession number 3BM8).
Consistent with the importance of Asp356 in binding of the
sulfonyl group in PVSN/PVS, an 11.7- and 9.7-fold decrease
in rate was observed for the PVSN-mediated inactivation of
the general acid deficient mutants YopH/D356A and YopH/
D356N. Taken together, the results indicate that efficient YopH
inactivation by PVSN/PVS requires both optimal hydrophobic
0.036 ( 0.004
0.163 ( 0.015
0.44 ( 0.06
LMW PTP
0.046 ( 0.008
and polar interactions between PVSN/PVS and YopH active
site, which are essential to placing the terminal vinyl carbon in
the immediate vicinity of Cys403 for nucleophilic attack.
Given the fact that the PVSN/PVS-mediated YopH inac-
tivation takes advantage of the common structural and
catalytic properties of the PTPs (e.g., pTyr binding pocket,
the P-loop, Cys403, Asp356, and Arg409), it is not surprising
that PVSN served as a irreversible time-dependent inactivator
for all PTPs tested, including cytosolic PTP1B and HePTP,
receptor-like DEP-1, the dual specificity phosphatases VHR
and PRL1, and the low molecular weight PTP (Table 2).
Furthermore, the ability of PVSN and PVS to engage the
PTP catalytic machinery should also impart specificity of the
vinyl sulfonates/sulfones toward the PTPs. Indeed, PVSN
failed to inhibit the reaction catalyzed by either acid or
alkaline phosphatases. PVSN was also 240-700-fold less
effective in inactivating the cysteine proteases papain and
cathepsin B than the mammalian PTPs HePTP and VHR
(Table S1). Further elaborations of the PVSN and PVS
scaffolds are expected to lead to the identification of aryl
vinyl sulfonates/sulfones with increased specificity toward
the PTPs.
Specificity of the PVSN-Based Probe in the Context of a
Complex Proteome. To determine whether the PVSN/PVS
scaffolds show specific labeling of the desired PTP in a complex
mixture, we employed a novel strategy for activity-based protein
profiling that utilizes the copper(I)-catalyzed azide-alkyne
cycloaddition reaction (“click chemistry”) to analyze the
functional state of enzymes in cell lystates.25 To this end, we
prepared an azide-tagged PVSN derivative, and we appended
an alkyne moiety onto biotin to facilitate click chemistry-
mediated capture of the PVSN-labeled PTPs from a complex
proteome (Scheme 1, and Materials and Methods). With these
reagents in hand, we assessed the compatibility of the PTP
labeling strategy in a well-defined system, namely E. coli, which
has no PTPs in its proteome. The specificity of the PVSN-based
probe for targeted PTP analysis was evaluated in total cell
lysates from both control E. coli and E. coli expressing
recombinant (His)6-tagged HePTP. Total cell lysates were
treated with the azide-tagged PVSN probe (20 µM) for 30 min
at room temperature and pH 6.0 and exposed to 0.125 mM of
the alkyne-labeled biotin, 1.25 mM TCEP, 0.127 mM TBTA,
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Scheme 1. Labeling of PTPs in Complex Proteomes with the
PVSN Probe by Click Chemistry
Figure 7. Labeling of HePTP in complex proteome by azide-tagged PVSN
probe and alkyne-labeled biotin. Total cell lysates with or without (His)6-
tagged HePTP were treated with the azide-tagged PVSN probe (20 µM)
for 30 min at room temperature and pH 6.0 and exposed to 0.125 mM of
the alkyne-labeled biotin, 1.25 mM TCEP, 0.127 mM TBTA, and 1.25 mM
CuSO4 for 1 h to conjugate the PVSN-adducted HePTP with biotin. The
identity of the (His)6-HePTP was revealed by its reactivity to anti-(His)6
antibody. PVSN labeling of HePTP was detected by antibiotin antibody.
and 1.25 mM CuSO4 for 1 h to conjugate the PVSN-adducted
HePTP with biotin for subsequent visualization. As shown in
Figure 7, Western-blot analysis of the reaction mixture with
antibiotin and anti-(His)6 antibodies showed robust labeling of
HePTP, while little background labeling was observed for the
entire non-PTP proteome under these conditions. This level of
labeling specificity indicates that the PVSN/PVS scaffolds can
be employed as activity-based probes to profile PTP activity in
complex proteome.
Advantages of PVSN and PVS as Mechanism-Based PTP
Probes. Compared to that of R-bromobenzyl phosphonate, there
are several advantages associated with the aryl vinyl sulfonate/
sulfone-based scaffolds. First, the kinetic parameters KI and ki
for the PVSN (0.29 mM and 0.061 min-1) and PVS (0.35 mM
and 0.080 min-1) mediated YopH inactivation compare very
favorably to those obtained for R-bromobenzyl phosphonate (4.1
mM and 0.11 min-1, respectively).14 The 12-14-fold higher
affinity exhibited by PVSN and PVS for the PTP active site
indicates that they are superior pTyr surrogates. Second,
R-bromobenzyl phosphonate undergoes significant solvolysis at
pH greater than 7,14 whereas PVSN and PVS are chemically
inert under similar conditions, even in the presence of 1 mM
glutathione (Figures S2 and S3). Third, like most pTyr mimetics,
R-bromobenzyl phosphonate is negatively charged and
exhibits exceedingly low membrane permeability. In contrast,
PVSN and PVS are neutral pTyr mimetics and are cell
permeable. Indeed, COS-7 cells treated with PVSN or PVS
showed increased global tyrosine phosphorylation profiles
comparable to that seen in vanadate (a commonly used potent
PTP inhibitor) treated cells (Figure 8). This result supports
the notion that PVSN and PVS can cross the plasma
membrane and inhibit PTP activity in vivo. In contrast, MVS,
an inactive analogue, and R-bromobenzyl phosphonate, which
is unable to penetrate the cell membrane, had little effect on
cellular tyrosine phosphorylation. Finally, unlike R-bro-
mobenzyl phosphonate, PVSN displayed a wide range of
intrinsic reactivity toward the PTPs, with kobs for HePTP
inactivation 30-fold greater than those for YopH, PTP1B,
DEP1, and low molecular weight PTP (Table 2). This
observation highlights the potential of developing isoform-
selective aryl vinyl sulfonate-based PTP probes for profiling
the activity of individual PTPs in signaling and in diseases.
Figure 8. COS-7 cells treated with PVS or PVSN display increased tyrosine
phonsphorylation. COS-7 cells were treated for 1 h with 0.5 and 1 mM
PVSN, PVS, MVS, and BBP or 1 mM sodium vanadate. Cell lysate (50-
µg protein) was resolved by 10% SDS-PAGE and transferred to nitrocel-
lulose membrane. Protein tyrosine phosphorylation level was determined
using anti-pTyr antibodies.
In summary, we reported kinetic and structural character-
ization of aryl vinyl sulfonates/sulfones as a new class of
mechanism-based probes of the PTPs. We showed that PVSN
and PVS inactivate a broad range of PTPs in a time- and
concentration-dependent fashion. We established that this
inactivation is active site-directed and irreversible. We
provided evidence that PVSN and PVS form covalent adduct
with the PTPs, arising from a Michael addition of the active-
site Cys Sγ onto the terminal carbon of the unsaturated vinyl
bond of PVSN/PVS. Our structural analysis of the YopH-
inactivator complexes revealed the molecular basis for the
mechanism and specificity of PVSN/PVS-mediated PTP
inactivation, which lies in the ability of PVSN and PVS to
take advantage of the conserved structural and catalytic
properties of the PTP active site. Specific binding interactions
between PVSN/PVS and the pTyr pocket position the
terminal vinyl carbon in the immediate vicinity of the active
site Cys for nucleophilic attack. Finally, we demonstrated
that, in contrast to the R-bromobenzyl phosphonate-based
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A R T I C L E S
Liu et al.
probes, PVSN and PVS are resistant to solvolysis and are
cell permeable and thus hold promise for in vivo applications.
Collectively, these properties bode well for the development
of aryl vinyl sulfonate/sulfone-based PTP probes to inter-
rogate PTP activity in complex proteomes.
Supporting Information Available: Details on the effect of
arsenate on the PVSN-mediated YopH inactivation, specificity
of PVSN toward the PTPs and non-PTP enzymes, and stability
of PVS in aqueous media and in the presence of glutathione.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Acknowledgment. This work was supported by National
Institutes of Health Grants CA126937 and CA69202.
JA711125P
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