Protein-tyrosine phosphatases (PTPs) are signal
transduction enzymes that catalyze the dephosphorylation of
phosphotyrosine residues via the formation of a transient
cysteinyl-phosphate intermediate. The mechanism of hydrolysis of this
intermediate has been examined by generating a Gln-262
Ala mutant
of PTP1B, which allows the accumulation and trapping of the
intermediate within a PTP1B crystal. The structure of the intermediate
at 2.5-Å resolution reveals that a conformationally flexible loop (the WPD loop) is closed over the entrance to the catalytic site,
sequestering the phosphocysteine intermediate and catalytic site water
molecules and preventing nonspecific phosphoryltransfer reactions to
extraneous phosphoryl acceptors. One of the catalytic site water
molecules, the likely nucleophile, forms a hydrogen bond to the
putative catalytic base, Asp-181. In the wild-type enzyme, the
nucleophilic water molecule would be coordinated by the side chain of
Gln-262. In combination with our previous structural data, we can now
visualize each of the reaction steps of the PTP catalytic pathway. The
hydrolysis of the cysteinyl-phosphate intermediate of PTPs is
reminiscent of GTP hydrolysis by the GTPases, in that both families of
enzymes utilize an invariant Gln residue to coordinate the attacking
nucleophilic water molecule.
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INTRODUCTION |
The formation of phosphoryl-enzyme intermediates is an essential
component of numerous enzymatic mechanisms that involve
phosphoryl-transfer reactions, for example the dephosphorylation of
Tyr(P) residues catalyzed by protein-tyrosine phosphatases (1).
PTPs1 together with the
protein-tyrosine kinases, which catalyze the opposing tyrosine
phosphorylation reaction, control the overall levels of cellular
tyrosine phosphorylation, and the molecular basis of the regulation and
substrate specificity of these enzymes is subject to much investigation
(2, 3). Reversible tyrosine phosphorylation is essential to the signal
transduction pathways triggered by hormones, mitogens, and oncogenes
that regulate such processes as cell growth, differentiation, and
proliferation. The PTPs are a diverse family of enzymes that comprise
transmembrane receptor-like PTPs (RPTPs) and soluble cytosolic
proteins. The catalytic domains of the PTPs are highly conserved,
consisting of ~250 amino acids and are characterized by an 11-residue
PTP signature motif, (I/V)HCXAGXXR(S/T)G,
containing the Cys and Arg residues that are essential for catalysis
(4, 5). Diversity within the family is generated by the nature of the
non-catalytic segments attached to the N and C termini of the PTP
domains, which provide regulatory and subcellular targeting functions.
Additional diversity is generated within the RPTPs, which frequently
possess tandem PTP domains, although for some RPTPs such as CD45, the C-terminal PTP domain lacks catalytic activity (3). The dual specificity phosphatases (DSPs) are related to the PTPs by their possession of the PTP signature motif and related tertiary structure and catalytic mechanism (1, 6).
Insights into the catalytic mechanism of PTPs and DSPs have been
obtained from structural and kinetic studies of these enzymes (1, 4,
5). A PTP1B-phosphotyrosine peptide complex revealed that the Tyr(P)
residue of the peptide is buried within a deep catalytic site cleft
present on the protein's molecular surface. The base of the catalytic
site is formed by residues of the PTP signature motif with the
phosphate group of Tyr(P) being coordinated by main-chain amide groups
and the Arg side chain of this motif, such that the phosphorus atom is
situated adjacent to the S
atom of the catalytic Cys residue (7).
Four other loops bearing invariant residues form the sides of the
catalytic cleft and contribute to catalysis and substrate recognition.
Engagement of phosphopeptides by PTP1B promotes a major conformational
change of one of these loops (the WPD loop) consisting of residues
179-187 that shift by as much as 8 Å to close over the phenyl ring of
Tyr(P) and allow the side chain of Asp-181 to act as a general acid in
the catalytic reaction. The Arg-221 side chain reorients to optimize salt bridge interactions with the phosphate bound to the catalytic site. This shift is coupled to motion of the WPD loop via a hydrogen bond between NH2 of Arg-221 and the carbonyl oxygen of
Pro-180 and hydrophobic interactions between the aliphatic moiety of
Arg-221 and the side chain of Trp-179. These interactions and the
hydrophobic packing between Phe-182 and the phenyl ring of Tyr(P)
stabilize the closed, catalytically competent conformation of the loop. The phosphotyrosine dephosphorylation reaction commences with nucleophilic attack by the S
atom of the catalytic cysteine on the
Tyr(P) phosphorus atom. Cleavage of the scissile P-O bond is
facilitated by protonation of the phenolic oxygen by Asp-181 with the
consequent formation of a phosphocysteine intermediate. The tyrosine
residue diffuses out of the catalytic site and subsequently the
transient phosphoryl-enzyme intermediate is hydrolyzed by an activated
water molecule. The structure of a PTP1B·WO4 complex suggests that after hydrolysis of the phosphoryl-enzyme intermediate, the WPD loop opens, allowing product release (8).
Numerous kinetic data support the reaction mechanism outlined above
(1). Cysteinyl-phosphate intermediates have been trapped by rapid
denaturation of PTPs and a DSP (vaccinia H1-related) during catalytic
turnover (9-11). Moreover, substitution of the catalytic Cys residue
for a serine abolishes catalytic activity and the formation of an
enzyme intermediate (9). The nucleophilicity of the Cys residue results
from its close proximity to main-chain amide groups and a hydrogen bond
with the side chain of Ser-222 of the PTP signature motif, resulting in
an unusually low pKa of 4.6 (12). The catalytic Asp
residue (Asp-181 of PTP1B) contributes to the basic limb of the pH
activity profile, and its substitution to Ala causes a
105-fold reduction in kcat,
suggestive of a role as an acid catalyst (13, 14). These results imply
that Asp-181 is necessary for the first step of the reaction, namely
cleavage of the Tyr(P) P-O bond and intermediate formation, a notion
consistent with the finding that Asp-181 mutants of PTP1B allow
phosphorylated substrates to form stable complexes with the enzyme
in vivo (14).
An understanding of the chemical steps of the phosphocysteine
dephosphorylation reaction has been more elusive, mainly because of the
transient character of this intermediate as PTPs are efficient catalysts. For example, PTP1B hydrolyzes 40 molecules of substrate(s)/s at 25 °C (13, 14). Of particular interest are the structure of the
cysteinyl-phosphate PTP intermediate, the mechanism of activation of
the nucleophilic water molecule responsible for the hydrolysis of the
intermediate, and mechanisms by which nonspecific phosphoryl-transfer
reactions are prevented. The phosphoryl transfer reaction to a water
molecule catalyzed by PTPs is highly specific, as PTPs are unable to
phosphorylate a range of primary alcohols and other phosphate acceptors
(15). Here, we describe an approach to obtain structural information on
the cysteinyl-phosphate intermediate of PTP1B. The structure of a
PTP1B-orthovanadate complex, which revealed that vanadate mimics the
pentavalent phosphorus transition state intermediate, indicated that
Gln-262 may play an important role in stabilizing and/or activating a
nucleophilic water molecule for attack on the cysteinyl-phosphate
intermediate. By replacing Gln-262 with Ala, we have prolonged the
life-time of the cysteinyl-phosphate intermediate, allowing us to trap
and visualize it using x-ray cryocrystallography. The structure (i)
reveals that the WPD loop adopts a closed conformation that sequesters
both the cysteinyl-phosphate residue and the nucleophilic water
molecule, (ii) suggests roles for Asp-181 and Gln-262 in catalyzing the
enzyme dephosphorylation reaction, and (iii) explains why phosphoryl
transfer from the intermediate occurs to water molecules and not to
other phosphoryl acceptors. The role of a Gln residue in catalyzing the
hydrolysis of the cysteinyl-phosphate intermediate of PTPs is
reminiscent of that performed by the Gln residue in GTP hydrolysis at
the catalytic site of GTPases (16-19). The approach that we describe here to trap the phosphoryl-enzyme intermediate of PTP1B may have general applicability to other transient covalent-enzyme intermediates and enzyme-substrate complexes that will allow their isolation and
structural characterization.
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EXPERIMENTAL PROCEDURES |
Preparation of Crystals--
Wild-type protein and the Q262A
mutant PTP1B protein and crystals were prepared as described (20). The
PTP1B-vanadate complex was prepared by incubating wild-type PTP1B
crystals in 100 mM HEPES (pH 7.5), 200 mM
magnesium acetate, 18% (w/v) polyethylene glycol 8000, and 1 mM sodium orthovandate for 10 min prior to transfer to the
same buffer with 25% (v/v) glycerol and freezing in a nitrogen gas
stream at 100 K (22). To prepare crystals of the
PTP1B-cysteinyl-phosphate complex, Q262A mutant crystals that had been
grown at pH 7.5 were incubated for 5 min in a buffer of 100 mM BisTris (pH 7.0), 200 mM magnesium acetate,
18% (w/v) polyethylene glycol 8000 and subsequently for another 5 min
in the same buffer at pH 6.5. To prepare for cryofreezing and for trapping the intermediate, crystals were incubated in this buffer with
5% and then 10% (v/v) methyl-pentanediol each for 5 min before being
transferred to 3 ml of 100 mM BisTris (pH 6.5), 200 mM magnesium acetate, 18% (w/v) polyethylene glycol 8000, 25 mM para-nitrophenol phosphate, 15% (v/v)
methyl-pentanediol for 12 min. At this time, crystals were mounted
directly into nylon loops and frozen.
Data Collection and Refinement--
Data for the intermediate
crystal were collected using a MAR 30-cm detector mounted on a Rigaku
x-ray generator, and data for the orthovanadate crystal were also
collected using a MAR 30-cm detector but on PX 9.6 at the Synchrotron
Radiation Source, Daresbury Laboratory. Both data sets were processed
using DENZO and SCALEPACK (21) (see Table
I for details). All refinement of the
coordinates was performed using X-PLOR (22) and the protein coordinates
and electron density maps analyzed with O (23) and TURBO-FRODO
(24).
Orthovanadate Complex--
Initial phases were derived from the
2.2-Å structure of PTP1B in an unliganded form to minimize model bias.
In this structure, no ligands were bound at the catalytic site and the
WPD loop adopts the open conformation. 2Fo
Fc and Fo
Fc OMIT maps (without orthovanadate in the model)
were calculated after 75 cycles of rigid body refinement, and these maps demonstrated density for the vanadate ion at the catalytic site, a
shift in the position of the WPD loop by 8 Å from the open to the
closed conformation, and a rotation of the Arg-221 side chain from an
all-trans to a cis conformation, indicative of
ligand binding. The model was subjected to 100 cycles of positional refinement, after which the coordinates for the WPD loop and Arg-221 side chain were rebuilt into the difference electron density. This
model was then subjected to simulated annealing with an initial temperature of 2500 K. A model for the orthovanadate ion was then constructed using perfect trigonal bipyramidal geometry and V-O bond
lengths of 1.61 Å. The ion was built into the difference density and
allowed to refine freely as a rigid body with interactions to the
protein turned off. The ion refined to a position 2.4 Å away from the
Cys-215 S
atom, consistent with the formation of a covalent bond
between the vanadium and Cys-215 S
atoms, and a bond of this length
was included in subsequent refinement. The model was then subjected to
further rounds of positional refinement and 15 cycles of individual
isotropic B-factor refinement. The bond length between the vanadium and
Cys-215 S
atom refined to a value of 2.4 Å both in the presence (at
200 kcal·mol
1) and absence of bond length constraints.
This value is similar to that reported by Denu et al. (25)
in their crystal structure of the Yersinia PTP-vanadate
complex. Water molecules were added manually with criteria for addition
being Fo
Fc density at 3
,
2Fo
Fc density at 1
, a
favorable hydrogen bonding environment, and a decrease in both the free and working R-factors after each cycle. The model was then
subjected to 50 cycles of positional refinement and 15 cycles of
B-factor refinement. Simulated annealing OMIT maps were calculated
after performing a simulated annealing refinement run (at 2000 K) using the refined protein and water coordinates and in the absence of the
vanadate coordinates. The final model satisfied or exceeded all
the stereochemical quality indicators of PROCHECK (26).
Cysteinyl-phosphate Intermediate--
Initial phases were also
calculated from the 2.2-Å structure of unliganded PTP1B. The model was
subjected to 75 cycles of rigid body and 100 cycles of positional
refinement, and 2Fo
Fc and
Fo
Fc OMIT maps (without intermediate) were calculated. The Fo
Fc difference map showed a 3
peak adjacent to the
Cys-215 S
atom and contiguous 2Fo
Fc density extending from the sulfur atom and
enveloping the difference density (Fig. 3A). The maps also showed movement of the WPD loop by 8 Å to the closed conformation and
a rotation of the Arg-221 side chain. Negative difference density was
also seen on the side chain of Gln-262 confirming the mutation. The
model was then subjected to simulated annealing from 2500 K. A model
for the phosphate intermediate was constructed with perfect tetrahedral
geometry around the phosphorus atom, P-O bond lengths of 1.55 Å, and
a P-S bond length of 1.85 Å. This model was built into the density,
the WPD loop was rebuilt into difference density, adopting the closed
conformation, and Gln-262 was mutated to Ala. The model was then
subjected to another 100 cycles of positional refinement and 15 cycles
of individual isotropic B-factor refinement. Water molecules were then
added manually, subject to the same criteria applied to the
PTP1B-vanadate complex, and the model was again subjected to positional
and B-factor refinement. A 2Fo
Fc electron density map using phases from refined
coordinates is shown in Fig. 3B. The final model satisfied
or exceeded all the stereochemical quality indicators of PROCHECK
(26).
 |
RESULTS |
Structure of a Transition State Analogue--
We investigated
initially the structure of the PTP1B-orthovanadate complex, which
provides information concerning the transition state structure of the
enzyme catalyzed Tyr(P) dephosphorylation reaction. A similar structure
of the distantly related Yersinia PTP has been reported
(25). Fig. 1A shows
2Fo
Fc and
Fo
Fc electron density maps
calculated using phases from a simulated annealing refinement run of
PTP1B coordinates excluding those for vanadate. A covalent bond is
formed between the S
atom of the nucleophilic Cys-215 residue and
the vanadium atom, so that the vanadate ion forms a pentavalent
trigonal bipyramidal structure that is an analogue of the pentavalent
transition state intermediate. At 2.3-Å resolution, we do not observe
distortion from perfect bipyramidal geometry. The three equatorial
oxygen atoms of vanadate form some eight hydrogen bonds to main-chain NH groups of the PTP signature motif and guanidinium side chain of
Arg-221, whereas the apical oxygen atom forms hydrogen bonds with the
side chains of Gln-262 and Asp-181 (Fig. 1B). The latter interaction suggests that the apical oxygen atom is protonated, forming
a hydrogen bond with the carboxylate group of Asp-181 at pH 7.5. The
apical oxygen atom most closely resembles the position of an attacking
nucleophilic water molecule during the hydrolysis step of the
cysteinyl-phosphate intermediate. Thus, the hydrogen bonds to Asp-181
and Gln-262 suggest that these residues may play a role in positioning
and/or activating a water molecule during this step. The interaction
between Asp-181 and the apical vanadate oxygen atom is possible as a
result of the closed conformation of the WPD loop, similar to that
observed in the PTP1B-phosphopeptide complex (7). Closure of the loop
is probably stabilized by a combination of interactions between Arg-221
and loop residues Trp-179 and Pro-180 and the hydrogen bond between
Asp-181 and the vanadate apical oxygen atom. A buried water molecule
(WAT1), analogous to that in the PTP1B·Tyr(P) complex, links the
main-chain amide groups of Phe-182 and Arg-221 with a vanadate
equatorial oxygen via a network of hydrogen bonds (Fig.
1B).

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Fig. 1.
A, simulated annealing OMIT
2Fo Fc (green) and
Fo Fc (red)
electron density maps of the PTP1B-vanadate complex superimposed onto
the refined coordinates. Phases and Fc coefficients
were calculated using protein coordinates (without those for vanadate)
from a simulated annealing refinement run. Contour levels are at 1
and 3 for the 2Fo Fc and
Fo Fc electron density maps,
respectively. This figure was generated using TURBO-FRODO (24).
B, refined structure of the PTP1B-vanadate complex. This
figure and all others except as indicated were generated using
X-OBJECTS (M. E. Noble, unpublished).
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Structure-based Design to Slow Rate of Phosphocysteine
Hydrolysis--
To understand the mechanism of cysteinyl-phosphate
hydrolysis more fully, we have sought to determine the structure of
this intermediate using x-ray crystallography. To achieve this, the rate of hydrolysis of the phosphoryl-enzyme intermediate should be
substantially slower than the rate of intermediate formation to allow
the intermediate to accumulate. Moreover, its life-time must be of
sufficient length to be amenable to structural analysis, being in
effect the rate-limiting step of the enzyme catalyzed reaction within
the crystal. The nature of the rate-limiting step catalyzed by PTPs and
DSPs is not entirely resolved. There are kinetic data to suggest that
the rate-limiting step of PTP catalysis is the rate of
phosphoryl-enzyme formation (25). This would imply that the
cysteinyl-phosphate intermediate would not accumulate appreciably
within the wild-type enzyme. However, other investigators, have
reported that the presence of a pre-steady state burst phase observed
during hydrolysis of para-nitrophenol phosphate
(p-NPP) indicates that hydrolysis of the phosphoryl-enzyme
intermediate is rate-limiting (27). The latter result implies that
during steady state catalysis, a phosphoryl-enzyme intermediate would accumulate, and this is consistent with the trapping, using rapid denaturation techniques, of phosphoryl-enzyme intermediates of PTPs
during the catalytic reaction (9-11, 25). However, since the
incorporation of phosphate in the phosphoryl-enzyme intermediate was
substoichiometric, the rates of phosphoryl enzyme formation and
hydrolysis are probably quite similar.
As discussed earlier, the structure of the PTP1B-vanadate complex
suggests crucial roles for Asp-181 and Gln-262, two invariant residues,
in positioning and/or activating the nucleophilic water molecule during
hydrolysis of the cysteinyl-phosphate intermediate. Recent kinetic data
have implicated Asp-92 of vaccinia H1-related (equivalent to Asp-181 of
PTP1B) as a catalytic base during phosphoryl-enzyme hydrolysis in
addition to its role as a catalytic acid to promote Tyr(P) cleavage
(25). We have reported previously that mutation of Gln-262 of PTP1B to
Ala reduces kcat by 100-fold and
Km 10-fold (13). In the PTP1B-phosphotyrosine
peptide complex, the position of Gln-262 differs from its position in
the PTP1B-vanadate complex, as the side chain is swung out of the
catalytic site to avoid a steric clash with the phenyl side chain of
the Tyr(P) substrate (7) (Fig. 2). This
implies that Gln-262 plays little or no catalytic function during
cleavage of the scissile P-O bond, whereas a role in positioning
and/or activating a water molecule during cysteinyl-phosphate
hydrolysis is likely. From this analysis, we reasoned that the 100-fold
reduction in kcat of the Q262A mutant may result
from reducing the rate of hydrolysis of the cysteinyl-phosphate intermediate without affecting the rate of enzyme phosphorylation. This
would render the rate-limiting step of the reaction as
phosphoryl-enzyme hydrolysis and increase the life-time of the
intermediate 100-fold. The wild-type enzyme kcat
is 40 s
1, whereas that of the Q262A mutant is ~0.5
s
1 at 25 °C (13). At 4 °C this corresponds to one
enzymatic reaction in ~10 s.

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Fig. 2.
Superimposition of the PTP1B-vanadate complex
and PTP1B·Tyr(P) complex in the vicinity of the catalytic site to
show conformational change of Gln-262. In the PTP1B·Tyr(P)
complex (cyan), Gln-262 is rotated out of the
catalytic site relative to the PTP1B-vanadate complex.
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Our approach to trapping the phosphocysteine intermediate was as
follows. By assuming that the rate of substrate diffusion into the
crystal exceeds the rate of cysteinyl-phosphate hydrolysis, and with an
excess of substrate added to the crystal incubation buffer, a steady
state accumulation of intermediate should be obtained, which may be
captured by flash-freezing a crystal in a stream of nitrogen gas at 100 K. The cysteinyl-phosphate intermediate is preserved in the frozen
crystals, which are suitable for x-ray data analysis. PTP1B crystals
are grown at pH 7.5 and 4 °C (20). However, at this pH, the rate of
PTP-catalyzed hydrolysis of p-NPP is 10% of its maximum
rate observed between pH 5.5 and 6.5 (27). The reduction in rate is
attributable in large part to the deprotonation of Asp-181, the
presumed acid catalyst in the p-NPP hydrolysis reaction,
which would imply that the rate-limiting step becomes that of enzyme
phosphorylation and hence a phosphoryl-enzyme intermediate would not
accumulate under these conditions (14, 27). Thus, to optimize the
conditions for enzyme phosphorylation, we equilibrated crystals at pH
6.5 prior to the start of the kinetic experiment. To start the
experiment, we incubated small crystals in a large molar excess
(~1000-fold) of p-NPP for 12 min at a high concentration (25 mM) to saturate the enzyme catalytic site to achieve
Vmax for enzyme phosphorylation. The
Km for p-NPP is ~1 mM (27).
Small crystals were used to optimize uniform distribution of substrate
within the crystal. Advantages of PTP1B crystals for this study are (i)
the high solvent content of 60%, which facilitates diffusion, and (ii)
the ability of small substrates such as Tyr(P) and p-NPP to
bind to the catalytic site and promote closure of the WPD loop (7). It
was assumed that, after 12 min, the kinetic reaction had reached steady
state and hence accumulation of the phosphoryl-enzyme intermediate had
been achieved. At this time, crystals were flash-frozen at 100 K. As a
control, we performed an identical experiment using wild-type PTP1B
crystals.
Structure of the Phosphocysteine Intermediate--
In the control
experiment, using the wild-type PTP1B crystals incubated with
p-NPP, no evidence for electron density corresponding to a
cysteinyl-phosphate intermediate or either substrate or product bound
to the catalytic site was observed. The WPD loop was in the open
conformation, reminiscent of the ligand free PTP1B state (8). The
absence of substrate is attributable to the rapid turnover of the
wild-type enzyme, and that of product, to the low phosphate product
concentration achieved at the time of crystal freezing.
However, for the PTP1B Q262A mutant, 2Fo
Fc and Fo
Fc electron density OMIT maps indicated that the
catalytic site Cys-215 residue had been modified to a phosphocysteine
residue representing the phosphoryl-enzyme intermediate (Fig.
3A). Clearly resolved density
was observed for three terminal oxygen atoms and for density bridging
the phosphorus atom of the phosphate with the S
atom of Cys-215. The
refined atomic temperature factors of the phosphate oxygen atoms are
between 30 and 33 Å2, higher than those for the oxygens of
the covalently bound vanadate ion, which are similar to the atoms of
Cys-215 (~16 Å2). The most likely explanations are
either an incomplete occupancy of the cysteinyl-phosphate residue
within the protein crystal or an increased mobility of the phosphoryl
group relative to the vanadate, which as a transition state analogue is
likely to be bound tightly. Electron density corresponding to three
water molecules interacting with the thiophosphate group is observed in
the 2Fo
Fc maps calculated
using structure factors from the refined protein coordinates (Fig.
3B). One of these (W1) is equivalent to WAT1 of the
PTP1B-vanadate complex and forms hydrogen bonds to the main-chain NH
groups of Phe-182 and Arg-221, the side chains of Arg-221 and Gln-266,
and a terminal oxygen of the cysteinyl-phosphate group (Fig.
4). A second water molecule (W2),
situated above the cysteinyl-phosphate group, participates in hydrogen
bonds with the side chain of Asp-181, W1, and a terminal oxygen atom of
the cysteinyl-phosphate. The position occupied by the Gln-262 side chain in the PTP1B wild-type enzyme is the site of a third water molecule (W3), which forms a hydrogen bond to the third
cysteinyl-phosphate oxygen atom.

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Fig. 3.
A, 2Fo Fc (green) and Fo Fc (red) electron density maps of the
PTP1B-cysteinyl-phosphate intermediate showing density bridging the
S atom of Cys-215 and phosphorus of the phosphoryl-enzyme
intermediate superimposed onto the refined coordinates. The maps were
calculated using phases derived from the apo-PTP1B complex (8) before
the coordinates for the cysteinyl-phosphate intermediate were included
in refinement and phase calculations. Contour levels are at 1 and
3 for the 2Fo Fc and
Fo Fc electron density maps,
respectively. B, 2Fo Fc (cyan) electron density map of the
PTP1B cysteinyl-phosphate intermediate showing catalytic site water
molecules. The maps were calculated using phases and
Fc coefficients derived from the refined
coordinates, and the electron density contour level is 1 .
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Fig. 4.
Refined structure of the
PTP1B-cysteinyl-phosphate intermediate in the vicinity of the catalytic
site.
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The overall protein conformation of the PTP1B-intermediate structure is
virtually identical with that of the wild-type PTP1B vanadate complex.
Equivalent atoms superimpose with a root-mean-square deviation of 0.8 Å. These overall similarities extend to the catalytic site residues of
the PTP signature motif (including the side chains of Cys-215 and
Arg-221). Within the resolution limits of the data, the terminal oxygen
atoms of the cysteinyl-phosphate intermediate superimpose almost
exactly onto the equivalent vanadate and phosphate oxygen atoms of the
PTP1B-vanadate and PTP1B·Tyr(P) complexes, respectively. Thus,
although a stereochemical inversion at the phosphorus atom occurs
during the phosphoryl-transfer reaction, the formation of a covalent
bond to the S
atom of Cys-215 shifts the center of mass of the
phosphoryl group toward the base of the catalytic site.
The WPD loop adopts the closed conformation that sequesters the
cysteinyl-phosphate intermediate and catalytic site water molecules
(Figs. 3 (A and B) and 4). The phenyl ring of
Phe-182, immediately C-terminal to the WPD motif, is positioned exactly 7 Å above the S
-P bond of the phosphocysteine intermediate. A small
difference between the wild-type PTP1B-vanadate complex and the
intermediate complex is that the carboxylate group of Asp-181 of the
intermediate complex is shifted by 1 Å away from cysteinyl-phosphate
group compared with its position in the vanadate complex (Fig.
5).

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Fig. 5.
Stereo view of a superimposition of
PTP1B-vanadate complex (cyan) and PTP1B-cysteinyl-phosphate
intermediate to show displacement of the putative nucleophilic water
molecule from in-line geometry of the S -P bond represented by the
apical oxygen of vanadate.
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DISCUSSION |
Implications for Catalytic Mechanism: Role of Asp-181 and Gln-262
and Catalytic Site Water Molecules--
During phosphocysteine
hydrolysis, an activated water molecule undergoes a nucleophilic attack
on the phosphorus atom of the intermediate. Such an attack is
kinetically unfavorable, as the phosphate terminal oxygen atoms
sterically repel an incoming nucleophile and, moreover, water molecules
exhibit low nucleophilicity (19). The structure of a PTP1B
phosphocysteine intermediate presented here provides an opportunity to
understand the mechanism of phosphocysteine hydrolysis. Critical
residues appear to be Asp-181 and Gln-262 and one of the buried water
molecules, W2. The position of this water molecule is reminiscent of a
water molecule (WAT3) observed in a Yersinia PTP-nitrate
(NO3) complex (28). Nitrate adopts a planar trigonal
geometry, mimicking the transition state during a phosphoryl transfer
reaction. In the Yersinia PTP·NO3 complex, WAT3 is positioned 4.2 Å above the nitrate nitrogen, in line with the
S
atom of the catalytic Cys-403 residue and hence well placed as a
nucleophile to attack a cysteinyl-phosphate intermediate. The water
forms hydrogen bonds to Gln-446 (equivalent to Gln-262 of PTP1B) and
Gln-357 of the WPD loop. A longer (3.3 Å) interaction is made with a
carboxylate oxygen of Asp-356 (equivalent to Asp-181 of PTP1B). Within
the PTP1B Q262A phosphocysteine complex, the position of W2 is
displaced by ~1.5 Å from being colinear with the Cys-215 S
-P bond
and the angle between the water molecule and S
-P bond is ~150°
(Fig. 4). The position of W2 in the PTP1B Q262A cysteinyl-phosphate
complex would be out of reach of hydrogen bonding to a Gln residue at
position 262 (4.2 and 4.5 Å, respectively, to OE1 and NE2 of Gln-262)
of wild-type PTP1B. This result provides a rationale for the reduced
rate of hydrolysis of the cysteinyl-phosphate intermediate of the PTP1B
Q262A mutant and suggests a plausible function for Gln-262 in
positioning the nucleophilic water molecule for attack on the
cysteinyl-phosphate intermediate. Model building shows that if W2 were
to form hydrogen bonds with Gln-262, as observed for the apical oxygen
atom of the PTP1B-vanadate complex, then the water molecule would be
ideally positioned for nucleophilic in-line attack on the
cysteinyl-phosphate group, being colinear with the P-S
bond and 3.5 Å from the phosphorus atom of the cysteinyl-phosphate. Thus, loss of
Gln-262 in the PTP1B Q262A mutant destabilizes the attacking water
molecule site and causes a shift to a position where in-line attack on
the cysteinyl-phosphate group is less favorable. An in-line position
for W2 would increase its distance to the carboxylate of Asp-181 (4.5 Å) if Asp-181 adopted the same conformation in the native
PTP1B-phosphocysteine complex as it does in the mutant PTP1B Q262A
phosphocysteine complex. Such as distance would not be favorable for
the Asp-181 to act as a general base. However, as noted above, the
position of the Asp-181 carboxylate group in the mutant PTP1B Q262A
phosphocysteine complex is shifted 1 Å out of the catalytic site
compared with its position in the wild-type PTP1B vanadate complex
(Fig. 5). A plausible model is that, in the wild-type PTP1B
phosphocysteine complex, the position of the Asp-181 carboxylate group
is similar to that in the PTP1B-vanadate complex. Such a position would
allow hydrogen bonds between W2 occupying an in-line attacking site
above the phospho-cysteine and simultaneous hydrogen bonds to the side
chains of Asp-181 and Gln-262. It is possible that in the PTP1B Q262A
phosphocysteine complex, loss of the Gln-262 residue removes the anchor
that stabilizes the position of the attacking water molecule (W2) above
the phosphocysteine and in-line with the P-S
bond. This shift of W2
to a position where it forms a hydrogen bond with one of the
phosphocysteine oxygen atoms causes a coupled shift of the position of
the Asp-181 side chain.
Conclusions and Perspectives--
The results that we describe
here, together with that of earlier work (1, 4, 5), allow each step of
the catalytic reaction of PTPs to be delineated in some detail,
outlined in Fig. 6 (A-E). The
focus of this investigation has been to understand the mechanism of
hydrolysis of the phosphocysteine intermediate. We show that in the
intermediate, the WPD loop adopts the closed, catalytically competent
conformation. This conformation serves two functions. The first is to
bring the side chain of the general base Asp-181 into the catalytic
site, and the second is to cap the catalytic site entrance, a role
played by the side chain of Phe-182, sequestering the phosphocysteine
intermediate and the buried water molecules and preventing
phosphoryl-transfer to extraneous phosphoryl acceptors. In contrast,
the coordination of a water molecule via a bidentate hydrogen bond to
Gln-262 favors specific phosphoryl transfer to a water molecule. The
phosphocysteine hydrolysis reaction can be described as follows. A
water molecule, coordinated by bifurcated hydrogen bonds to the side
chain of Gln-262 and a single hydrogen bond with Asp-181 is situated
directly above and in-line with the P-S
bond of the
phosphocysteine. The relative pKa values of Asp and
Gln would suggest that it is most likely that the role of Gln-262 is to
position the water molecule correctly, and that of Asp-181 to function
as a general base. Abstraction of a proton from the water by Asp-181
increases the nucleophilicity of the water molecule, facilitating
attack onto the phosphorus atom of the intermediate. Dixon and
colleagues (25) have proposed that the negative charge that develops on the S
atom of Cys-215 during the phosphoryl-displacement is
compensated by a hydrogen bond to the hydroxyl group of the Ser/Thr
residue within the PTP motif (Fig. 4). This is consistent with the
invariance of a either a Ser or Thr at this position within the PTP
family and the finding that substitution to Ala reduces the rate of
phosphocysteine hydrolysis 100-fold without affecting the rate of
Tyr(P) P-O bond cleavage (25, 29). In our structure, we observe a
hydrogen bond between the S
atom of the cysteinyl-phosphate residue
and OH of Ser-222, supporting this mechanism (Fig. 4). Where
displacement reactions are dissociative rather than associative in
character, considerable rate enhancement is achieved when the negative
charge that develops on the leaving group is compensated. Less
important for catalysis is the need to activate the attacking
nucleophilic species (19). Thus, the observation that substitution of
an Ala for the Ser/Thr residue within the PTP motif affects only the
rate of cysteinyl-phosphate hydrolysis and not its rate of formation
(25, 29) may be explained by a model where the two reaction steps
catalyzed by PTPs are both dissociative in character. A schematic of
the reaction mechanism is shown in Fig.
7. Our model for the role of Gln-262 is
supported by the findings that substitution of the equivalent residue
in the Yersinia PTP for Ala reduces the steady state but not
the burst phase rate, and moreover, allows phosphoryl-transfer to
alcohols (15).

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Fig. 6.
Stereo views depicting the sequences of the
reaction pathway catalyzed by PTP1B. A, PTP1B apoenzyme,
with WPD loop open. B, PTP1B C215S Tyr(P)-Michaelis complex,
with WPD loop closed; Gln-262 swings out of the catalytic site.
C, PTP1B Q262A cysteinyl-phosphate intermediate complex,
with WPD loop closed. D, PTP1B vanadate complex,
representing the transition state of cysteinyl-phosphate hydrolysis,
with WPD loop closed; Gln-262 swings back into the catalytic site.
E, PTP1B tungstate-product complex, with WPD loop
open.
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Fig. 7.
Schematic of the reaction mechanism catalyzed
by PTP1B. A, formation of the cysteinyl-phosphate
intermediate. B, hydrolysis of the cysteinyl-phosphate
intermediate.
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The reactions catalyzed by PTPs share many features in common with that
of GTP hydrolysis by the GTPases. For example, the role of Gln-262 in
positioning a water molecule for nucleophilic attack onto the
phosphocysteine intermediate of PTP1B is reminiscent of the Gln residue
at the catalytic site of most GTPases, including the Ras family and
G
subunits of the heterotrimeric G-proteins. Mutation of the
catalytic site Gln-61 residue in Ras causes cellular transformation
(30) and a 10-fold reduction in the rate of GTP hydrolysis (31, 32).
Similarly, mutations of the equivalent Gln residue within the catalytic
sites of G
s and G
i reduce the intrinsic
rate of GTP hydrolysis and are associated with thyroid and pituitary
tumors (33, 34). Crystal structures of complexes of G
t
and G
i with GDP, Mg2+, and
AlF4
(16, 17) and a recent structure
of a Ras-RasGAP, GDP, Mg2+, AlF3 complex (18)
have revealed the active site conformations of these enzymes and the
mechanisms of GTP hydrolysis. Gln-61 (and its homolog in the
heterotrimeric G-proteins) plays a dual role in catalyzing GTP
hydrolysis. First, it coordinates the attacking water molecule and
positions it optimally for in-line approach onto the GTP-
-phosphate.
Second, it forms a hydrogen bond with the
-phosphate oxygen atom as
it passes through the transition state, stabilizing the pentavalent
phosphorus transition state. In PTP1B, Gln-262 also coordinates the
attacking water molecule to position it for in-line attack, although,
unlike GTPase, Gln-262 does not hydrogen-bond to the oxygens of the
cysteinyl-phosphate. In addition to a common Gln residue, both PTPs and
GTPases utilize an essential Arg residue that coordinates and
stabilizes the pentavalent phosphorus intermediate (16-18,
35).
In conclusion, we have succeeded in trapping the PTP1B
phosphoryl-enzyme intermediate by introducing a catalytic site mutation to reduce the rate of cysteinyl-phosphate hydrolysis, rendering this
step as the rate-limiting step of the PTP catalyzed reaction. In
combination with our earlier work (7, 8), this allows us to delineate
each step of the reaction pathway.