Insights into the reaction of protein-tyrosine phosphatase 1B: Crystal structures for transition state analogs of both catalytic steps

Article abstract of DOI:10.1074/jbc.M109.066951

Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal

Full text of DOI:10.1074/jbc.M109.066951

Protein Structure and Folding:
Insights into the Reaction of
Protein-tyrosine Phosphatase 1B:
CRYSTAL STRUCTURES FOR
TRANSITION STATE ANALOGS OF
BOTH CATALYTIC STEPS
Tiago A. S. Brandão, Alvan C. Hengge and
Sean J. Johnson
J. Biol. Chem. 2010, 285:15874-15883.
doi: 10.1074/jbc.M109.066951 originally published online March 16, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 21, pp. 15874–15883, May 21, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Insights into the Reaction of Protein-tyrosine Phosphatase 1B
□
S
CRYSTAL STRUCTURES FOR TRANSITION STATE ANALOGS OF BOTH CATALYTIC STEPS*
Received for publication, September 17, 2009, and in revised form, March 3, 2010 Published, JBC Papers in Press, March 16, 2010, DOI 10.1074/jbc.M109.066951
Tiago A. S. Branda˜o1, Alvan C. Hengge2, and Sean J. Johnson3
From the Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300
Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs
through a two-step mechanism involving a phosphocysteine
intermediate. We have solved crystal structures for the transi-
tion state analogs for both steps. Together with previously
reported crystal structures of apo-PTP1B, the Michaelis com-
plex of an inactive mutant, the phosphoenzyme intermediate,
and the product complex, a full picture of all catalytic steps can
now be depicted. The transition state analog for the first cata-
lytic step comprises a ternary complex between the catalytic cys-
teine of PTP1B, vanadate, and the peptide DADEYL, a fragment
of a physiological substrate. The equatorial vanadate oxygen
atoms bind to the P-loop, and the apical positions are occupied
by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur
atom. The vanadate assumes a trigonal bipyramidal geometry in
both transition state analog structures, with very similar apical
O–O distances, denoting similar transition states for both phos-
phoryl transfer steps. Detailed interactions between the flank-
ing peptide and the enzyme are discussed.
is believed to suppress potential tumors by antagonizing signal-
ing of oncogenic factors (9, 10). Other PTPs are known viru-
lence factors for a number of human diseases (11–13).
Reactions catalyzed by PTPs take place by a ping-pong mech-
anism (Fig. 1) (2). In the first step, a nucleophilic cysteine thio-
late attacks the phosphate ester moiety of the substrate, result-
ing in formation of a phosphoenzyme intermediate with release
of the peptidyl tyrosine. The second step occurs via attack of
water on the phosphoenzyme intermediate and yields the final
products inorganic phosphate and the regenerated enzyme.
The central binding site for the substrate is the P-loop, a region
at the bottom of a pocket that includes the nucleophilic cysteine
and backbone amide groups oriented in a horseshoe fashion.
The amide protons in the P-loop, together with a conserved
arginine residue, hydrogen-bond to the phosphoryl group of
the substrate and orient it for nucleophilic attack, providing
transition state (TS) stabilization. Substrate binding is followed
by conformational changes that culminate with closure of the
active site pocket by a conserved and flexible loop of sequence
WPD. The aspartic acid in this loop functions as a general acid
in the first step and as a general base catalyst in the second step.
The design of PTP inhibitors is a very active area of research.
A common strategy is focused on the synthesis of competitive
inhibitors that block the active site (6, 11, 14). Binding affinities
in these cases range from millimolar to micromolar, and spec-
ificity is often compromised by the high similarity between PTP
active sites. In order to achieve more potency and selectivity,
more extensive interactions are achieved by using doubly tar-
geted inhibitors (9). For example, in PTP1B, targeting of a sec-
ond Tyr(P) binding site in the vicinity of the active site has been
used. In such cases, inhibition constants can be improved to the
nanomolar range (15), and selectivity is gained because the sec-
ond Tyr(P) site in PTP1B is not conserved among all PTPs.
One important reason for the limited success of single site
inhibition in PTPs is the fact that substrate binding is far sur-
passed by the affinity of the TS. Like other PTPs, PTP1B cata-
lyzes the hydrolysis of phosphate ester dianions, which have
very slow rates of uncatalyzed hydrolysis (e.g. the half-life for
the uncatalyzed attack of water on the dianion of alkyl phos-
phate esters is ϳ1.1 ϫ 1012 years (k ϭ 2 ϫ 10Ϫ20 sϪ1 at 25 °C))
(16). Catalytic efficiency is achieved by selective affinity for
the TS relative to the substrate. According to the TS binding
paradigm, TS affinity is equivalent to the ratio between the
specificity constant (kcat/Km) for the catalyzed reaction and its
uncatalyzed rate constant (17). Based on kinetic constants for
catalyzed (18) and uncatalyzed (16) reactions (see supplemen-
The phosphorylation of tyrosine residues by protein-tyrosine
kinases and the reverse action by protein-tyrosine phospha-
tases (PTPs)4 is a common mechanism for the control of
biological pathways (1–3). Protein-tyrosine phosphatase 1B
(PTP1B) is a biomedically important phosphatase with several
roles, including negative regulation of insulin signaling by
dephosphorylation of the insulin receptor tyrosine kinase (4).
Knock-out studies show that loss of PTP1B is associated with
an increased insulin sensitivity and suppression of weight gain
in mice (5). As a result, this enzyme has been considered a
significant target for treatment of type 2 diabetes and obesity (6,
7). PTP1B also down-regulates cell growth by dephosphorylat-
ing the epidermal growth factor receptor (8). Overexpression
has been observed in human breast and ovarian cancer, where it
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant GM47297 (to A. C. H.).
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Table S1 and Figs. S1–S5.
The atomic coordinates and structure factors (codes 3I7Z and 3I80) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
1 RecipientofafellowshipfromCoordenac¸a˜odeAperfeic¸oamentodePessoal
de Nível Superior (Brazil).
2 To whom correspondence may be addressed. Tel.: 435-797-3442; Fax: 435-
797-3390; E-mail: alvan.hengge@usu.edu.
3 To whom correspondence may be addressed. Tel.: 435-797-2089; Fax: 435-
797-3390; E-mail: sean.johnson@usu.edu.
4 The abbreviations used are: PTP, protein-tyrosine phosphatase; TS, transi-
tion state; pNPP, p-nitrophenyl phosphate; KIE, kinetic isotope effect;
bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
MC, Michaelis complex; PDB, Protein Data Bank.
tal material), PTP1B exhibits transition state (TS) affinities of
Ϫ1
Ͼ1013
M
Ϫ1 for p-nitrophenyl phosphate (pNPP) and 1019
M
15874 JOURNAL OF BIOLOGICAL CHEMISTRY
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The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
FIGURE 1. The general mechanism of the PTP-catalyzed reaction.
when the substrate is the peptide acetyl-DADEpYL-NH2. (Arg221) involved in binding and transition state stabiliza-
Besides the known closure of the WPD-loop, the existence of tion; 2) the WPD-loop (residues 179–187), bearing the gen-
other catalysis-associated conformational changes that might eral acid/base (Asp181), which is active when this loop alter-
affect protein-substrate interactions is uncertain.
nates from the open to the closed conformation; 3) the
In order to better understand structural changes associated Q-loop (residues 261–262), including the residue Gln262, an
with catalysis, we have crystallized transition state analogs for important player in the second catalytic step; 4) the lysine
the first and second catalytic steps. Kinetic isotope effects loop (residues 119–121), containing Lys120, a conserved res-
(KIEs) for the reaction of p-nitrophenyl phosphate by PTP1B idue involved in key interactions with the WPD-loop (31);
were measured to verify that its transition state structure is and 5) the Tyr(P) recognition loop (residues 47–49),
similar to those of other PTPs. The first step TS analog reported involved in substrate recognition and binding.
here is a ternary complex that involves a central metavanadate
EXPERIMENTAL PROCEDURES
with two apical ligands, the nucleophilic cysteine thiolate and
the phenol leaving group, the latter as the tyrosyl oxygen atom
The expression and purification of wild type PTP1B were
in the peptide sequence DADEYL. This peptide is a fragment of slightly modified from previously reported methods (32) (see
the epidermal growth factor receptor, which, in phosphory- supplemental material). Protein concentrations were moni-
280 nm
lated form, exhibits strong affinity for PTP1B (18). A previous tored by UV using an A
ϭ 1.24. The peptide DADEYL
1 mg/ml
crystallographic study shows a Michaelis complex between a (Ͼ98% pure) was a custom synthesis by Anaspec. Labeled and
catalytically inactive C215S mutant of PTP1B and the phos- natural abundance pNPP (dicyclohexylammonium salt) were
phopeptide DADEpYL (19). The second step TS analog re- synthesized according to previous methods (33). Sodium
ported here is a complex between PTP1B and orthovanadate. orthovanadate (Na3VO4) was purchased from Fisher.
Although a related structure has been previously described
Kinetic Isotope Effect Determinations—KIEs were measured
(18), the coordinates are not publicly available, and it was there- using the internal competition method and thus are isotope
fore necessary to determine a new structure approximating the effects on V/K (34). In the commonly used notation, a leading
second transition state. Comparisons of these newly deter- superscript of the heavier isotope is used to indicate the isotope
mined and previously reported structures highlight significant effect on the following kinetic quantity (e.g. 15(V/K) denotes
protein conformational changes arising during catalysis.
These TS analogs are based on vanadate ions, which are tet-
14(V/K)/15(V/K), the nitrogen-15 KIE on V/K).
Natural abundance pNPP was used for measurements of
rahedral in solution but in active sites of PTPs form stable pen- 15(V/K). The 18O KIEs 18(V/K)bridge and 18(V/K)nonbridge were
tavalent structures exhibiting trigonal bipyramid geometries measured by the remote label method, using the nitrogen atom
(18, 20–23). Such structures are widely accepted as sharing in p-nitrophenol as a reporter for isotopic fractionation in the
close structural characteristics with the metaphosphate-like labeled oxygen positions (35). The isotopic isomers used are
transition state in phosphate transfer (24). In comparison with shown in the supplemental material. Isotope effect determina-
Ϫ
Ϫ
other analogs (25–27), such as WO24Ϫ, MgF3 , and NO3 , com- tions were made at 25 °C in 50 mM bis-tris buffer, pH 5.5, con-
plexes based on vanadate have the advantage of displaying the taining 1 mM dithiothreitol. The pNPP concentration was 18
same overall charge as phosphate and can easily derivatize and mM, and the reaction was started by the addition of wild type
interact with ligands at their axial positions.
PTP1B to a final concentration of 0.21 ␮M. Within 15–20 min,
The new TS analog structures, together with previously the reactions reached 40–60% completion and were stopped by
solved structures of the apoenzyme (28), phosphoenzyme titrating with HCl to pH 2–3. Protocols for isolation of p-nitro-
intermediate (29), and product complex (30), now provide a phenol and isotopic analysis and calculation of the isotope
depiction of all of the catalytic steps involved in the mechanism effects were the same as previously described (36) and are
of PTP1B. This full array of structures permits a step-by-step described in the supplemental material.
description of the structural changes involved in binding and
Crystallization—Crystals were grown using sitting drop
catalysis, particularly those related to the following protein vapor diffusion at 4 °C by mixing 2 ␮l of protein solution, 0.5 ␮l
segments: 1) the P-loop (residues 215–222), which includes of 30% (w/v) sucrose, and 3 ␮l of precipitant solution (0.1 M
the nucleophilic cysteine (Cys215) and an important serine Hepes, pH 7.5, 0.2 M magnesium acetate, and 15–17% polyeth-
(Ser222) that stabilizes its thiolate form, and the arginine ylene glycol 8000). Single crystals appeared after 3 days. The
MAY 21, 2010•VOLUME 285•NUMBER 21
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The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
TABLE 1
protein solution was prepared as follows. 0.36 ␮l of 100 mM of
Data collection and refinement statistics for the PTP1B transition
state analogs
Values in parentheses correspond to those in the outer resolution shell.
Na3VO4 and 10 ␮l of 50 mM DADEYL peptide (at pH 8.5–9.0)
were mixed and allowed to react for 1–1.5 h; 50 ␮l of native
PTP1B (12 mg/ml in 10 mM Tris, pH 7.5, 25 mM NaCl, 0.2 mM
EDTA, and 3 mM dithiothreitol) was then added to the peptide/
vanadate mixture and immediately placed in the crystallization
tray. The final protein solution contained a ratio of 1:3:30 be-
tween PTP1B, Na3VO4, and the peptide DADEYL. Crystalliza-
tion at higher concentrations of Na3VO4 resulted in different
crystal morphology; a 30:3 vanadate/peptide ratio gave crystals
that proved to be a binary complex between PTP1B and
VO34Ϫ. Cryoprotection was performed by transferring crystals
stepwise into stabilization solution with increasing glycerol
amounts to a final concentration of 15% and the respective ini-
tial concentrations of ligands present in the protein solution,
and then flash-cooled in liquid nitrogen.
PTP1B-VO3-DADEYL
PTP1B-VO4
(TSA1)
(TSA2)
Data collection
Beamline
Home source
1.5418
Home source
1.5418
Wavelength (Å)
Resolution range (Å)
Outer shell (Å)
No. of reflections
Unique
29.43–2.30
2.38-2.30
38.04–2.25
2.33-2.25
24,185
135,032
22,453
89,995
Total
Average redundancy
Mean I/␴(I)
5.6 (5.6)
4.0 (4.0)
9.2 (2.3)
8.6 (2.2)
Completeness (%)
99.9 (100.0)
7.5 (54.0)
P3121
100.0 (99.9)
8.4 (47.3)
P3121
R
sym (%)a
Space group
Unit cell dimensions (a, b, c (Å);
88.0, 88.0, 118.6
87.8, 87.8, 103.8
␣ ϭ ␤ ϭ 90° and ␥ ϭ 120°)
Refinement
R
work/Rfree (%)b
20.6/23.2
19.9/23.5
Data Collection, Structure Determination, and Refinement—
Diffraction data were collected using a home source generator
and detector (Rigaku RU-200/Raxis IVϩϩ). Data were indexed
and processed using d*TREK in the program Crystal Clear (37).
Molecular replacement was performed using Phaser (38) from
the CCP4 program suite (39, 40). The search model was the apo
wild type PTP1B structure (Protein Data Bank (PDB) entry
2CM2) with the active site water molecules removed. Refine-
ment was performed using the program Phenix (41). The TLS
groups were generated by the TLS Motion Determination
server (42). Coot (43) and MolProbity (44) were used for model
building and validation. The crystallographic data and statistics
of structure refinement are given in Table 1. The observed dif-
ferences in B-factors between TSA1 (transition state analog 1)
and TSA2 (TSA1 Ͼ TSA2) are due to tighter crystal packing
along the c axis of the unit cell in TSA2. The bound peptide in
TSA1 partially disrupts packing along the c axis, resulting in
increased spacing and conformational flexibility between adja-
cent molecules. Structure factors and coordinates for the
PTP1B-VO3-DADEYL and PTP1B-VO4 structures have been
deposited in the PDB under accession numbers 3I7Z and 3I80,
respectively. All figures depicting crystal structures were pre-
pared using PyMOL (45).
Atoms in the structure
Protein
2482
150
28
2440
231
31
Waters
Ligands/ions
Average B-factors (Å2)
Protein
75.3
66.8
44.4
53.0
Water
Ligands/ions
100.1
64.4
Root mean square deviation
bond (Å)/angle (degrees)
Protein geometryc
Ramachandran outliers (%)
Ramachandran favored (%)
Rotamer outliers (%)
Protein Data Bank entry
0.009/1.0
0.006/0.9
0.3
98.0
0.0
0.3
98.3
0.4
3I7Z
3I80
a Rsym ϭ (͚͉(I
Ϫ ͗I͘)͉)/(͚I), where ͗I͘ is the average intensity of multiple
measurements.
b Rwork ϭ (͚͉Fobs Ϫ Fcalc͉)/(͚͉Fobs͉) and is calculated using all data; Rfree is the R-fac-
tor based on 5% of the data excluded from refinement.
c Ramachandran statistics were calculated using the MolProbity server (44).
TABLE 2
Isotope effects for the catalyzed reaction of pNPP by the wild type
enzymes PTP1B, PTP1, and YopH and the YopH general acid mutant
All were measured at the pH optimum for activity. Values in parentheses after KIE
data are S.E. in the last decimal place.
Reaction
15(V/K)
18(V/K)bridge
18(V/K)nonbridge
1.0018(5)
0.998(2)
1.000(1)
1.0007(5)
PTP1B wild typea
1.0004(2)
1.0001(2)
0.9999(3)
1.0022(3)
1.0121(9)
1.0142(4)
1.0152(6)
1.0274(8)
Rat PTP1a (36)
YopH wild typeb (36)
YopH D356Ac (36)
a pH 5.5 and 25 °C.
b pH 5.0 and 25 °C.
c pH 6.0 and 35 °C.
RESULTS
Kinetic Isotope Effects—Reactions of wild type PTP1B with
pNPP were carried out at the optimum pH of 5.5 and 25 °C, composite omit map of the active site revealed electronic den-
where Km ϭ 0.58 Ϯ 0.01 mM and kcat ϭ 24.4 Ϯ 0.4 sϪ1. The sity for a single vanadate, exhibiting a trigonal bipyramidal
isotope effect data are shown in Table 2. As the enzymatic structure conforming to TSA2, and the map showed no evi-
substrate of PTPs is the dianionic form of pNPP, the 18(V/ dence for a bound peptide. When the peptide/vanadate ratio
K)nonbridge isotope effect was corrected considering the iso- was 30:3, crystals grew with hexagonal bipyramidal morphol-
tope fractionation for protonation of the nonbridge oxygen ogy. Composite omit maps for the active site reveal clear elec-
atoms because pNPP is present as a mixture of monoanion tronic density for the vanadyl-peptide complex and a trigonal
and dianion forms at this pH. Data for the reaction catalyzed bipyramid structure that is consistent with TSA1. Supple-
by the other PTPs YopH from Yersinia and rat PTP1 are also mental Fig. S2 displays composite omit maps for residues in the
shown (36) for comparison purposes.
active site of TSA1 and TSA2.
Crystallography—The two transition state analogs were crys-
Fig. 2 shows key active site interactions in the TS analog
tallized under similar conditions, except for the concentrations structures. The apical positions of the vanadate moieties of
of peptide and vanadate, which were optimized to give the both TSA1 and TSA2 are occupied by the sulfur of Cys215 and
structures of the transition state analog for the first step (TSA1) by an oxygen atom at distances of 2.5 and 2.1 Å, respectively,
and the second step (TSA2). With a ratio of peptide/vanadate/ from the central vanadium atom. The equatorial oxygen atoms
enzyme of 3:30:1, hexagonal prism crystals were obtained. The hydrogen-bond to backbone amide groups of the P-loop and
15876 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 21•MAY 21, 2010

The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
the guanidinium group of Arg221
,
which is rotated in relation to the
apo structure by about 82° around
the ␹-3 dihedral angle (see Fig. 3).
Substrate and competitive inhibi-
tors cause the same conformational
change in Arg221 (19, 28, 46). In this
conformation, the side chain of
Arg221 hydrogen-bonds with the
backbone carbonyl of Trp179 and
assists the WPD-loop in a shift from
the open, catalytically inactive to
the closed, active conformation.
This brings the WPD-loop residue
Asp181 into proximity to protonate
the leaving group oxygen (47). The
essential role of this conserved argi-
nine in facilitating WPD-loop clo-
sure has been shown in the closely
related PTP YopH, where the
R221K mutation disables general
acid catalysis, presumably by affect-
ing WPD-loop movement (33).
FIGURE 2. Orientation of key residues at the active site of PTP1B and the binding environment for trigonal
bipyramid structures in the transition state analogs. a, first transition state complex (TSA1) between native
PTP1B, metavanadate, and the Tyr in the peptide DADEYL; b, second transition state (TSA2) complex between
native PTP1B and orthovanadate. The Trp179 ring was omitted for the sake of clarity, and only the backbone
carbonyl group is shown. Hydrogen bond distances (in red) are in Å. See supplemental Fig. S2 for electron
density surrounding residues at the active site of TSA1 and TSA2. Stereo images of a and b are included in
supplemental Fig. S3.
Gln262 in TSA1 occupies a similar
position as in the Michaelis complex
and is rotated 132° counterclock-
wise in relation to TSA2 (Fig. 2b).
This residue is believed to position a
nucleophilic water molecule in the
second catalytic step (29). In TSA1,
the amide proton of the side chain
of Gln262 hydrogen bonds to the
carbonyl oxygen of Gly259 (not de-
picted). The same interaction is
observed in the apo and Michaelis
complex. In TSA2, the side chain of
Gln262 rotates into the active site,
and the amide carbonyl oxygen
hydrogen-bonds to the apical oxy-
gen in the trigonal bipyramidal van-
adate moiety.
In both TSA1 and TSA2 struc-
tures, Lys120 adopts a similar con-
formation, hydrogen-bonding to
Asp181 and Glu115 at donor-accep-
tor distances of 2.6 and 2.7 Å,
respectively. In the Michaelis com-
plex, distances are 2.8 and 3.0 Å,
respectively. In contrast, in the
phosphoenzyme, the Lys120 side
chain does not hydrogen-bond to
other protein residues. This residue
has been suggested to be part of
dynamic events during substrate
and inhibitor binding (31, 48). The
K120A mutation has little effect on
Km and kcat for pNPP hydrolysis and
Ki values for vanadate (49). How-
FIGURE 3. Crystal structures along the pathway of the reaction catalyzed by PTP1B. Hydrogen bond
distances are in Å. The Trp179 ring was omitted for the sake of clarity, and only the backbone carbonyl
group is shown. a, resting state PTP1B apoenzyme (PDB entry 2CM2) (28); b, Michaelis complex between
PTP1B C215S and the peptide DADEpYL (PDB entry 1PTU) (19); c, first transition state complex between
native PTP1B, metavanadate, and the Tyr in the peptide DADEYL (new structure, PDB entry 3I7Z); d, PTP1B
Q262A cysteinyl-phosphate intermediate enzyme (PDB entry 1A5Y) (29); e, second transition state com-
plex, between native PTP1B and orthovanadate (new structure, PDB entry 3I80); f, PTP1B-tungstate prod-
uct analog complex (PDB entry 2HNQ) (30). These structures are further summarized in supple-
mental Table S1.
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The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
ever, the use of a nonbiological substrate may preclude a proper number of enzymatic and uncatalyzed phosphate ester reac-
definition of the role of this residue. In fact, protein alignments tions (35, 53–57), which provides a background for the inter-
reveal that most PTPs retain a Lys or Arg at this position, sug- pretation of the KIE data measured in this work.
gesting an important role for this residue (50). The crystal
The three KIEs give different information regarding transi-
structure of a complex between PTP1B and insulin receptor tion structure and charge. The magnitude of 15(V/K) reflects
tyrosine kinase reveals that Lys120 is involved in a salt bridge the amount of negative charge on the leaving group in the tran-
interaction with insulin receptor tyrosine kinase (51).
sition state. This KIE results from delocalization of charge aris-
Extensive interactions occur between the peptide of TSA1 ing from the P–O bond fission, which involves the nitrogen via
and a surface groove adjacent to the active site (supplemental resonance. This KIE reaches a maximum of ϳ1.003 for transi-
Fig. S4). These interactions include hydrogen bonds between tion states with extensive P–O bond fission and no charge neu-
the backbone amide proton of Arg47 in PTP1B and the back- tralization (35). The negligible 15(V/K) observed indicates that
bone carbonyl oxygen of AspϪ2 in the peptide and between the efficient charge neutralization occurs in the transition state.
side chain carboxyl of Asp48 in PTP1B and the backbone amide The 18(V/K)bridge is a measure of P–O bond fission. A large
ϩ1
protons of Tyr(P) and Leu in the peptide.5 Similar interac- normal value indicates extensive P–O bond weakening and can
tions are observed in the Michaelis complex along the core of reach 1.03 for a loose transition state with extensive bond fis-
the peptide, although positions of the peptide backbone and sion (35). The 18(V/K)bridge KIE also reflects an inverse contri-
N-terminal residue side chains are slightly different. The bution from protonation. The equilibrium isotope effect for
change in position of the peptide backbone is accompanied by protonation of nitrophenol is 0.985 (58), so a transition state
movement of the aryl ring of the substrate, which is about 0.45 where P–O fission and protonation are both far advanced
Å farther away from the active site in TSA1 than in the Michae- would show a KIE of about 1.015. PTP reactions show 18(V/
lis complex, as measured by the distance between the aryl ring K)bridge KIEs close to this value. The secondary isotope effect
and C␣ of Arg221. Differences in N-terminal regions of the pep- 18(V/K)nonbridge reveals changes in bonding to the phosphoryl
tides may arise from crystal packing. Hydrogen bonds along this group (metaphosphate-like or phosphorane-like) in the transi-
region are observed with adjacent protein molecules; in TSA1, tion state. This KIE is small and inverse for metaphosphate-like
Ϫ4
the side chain of Asp hydrogen-bonds to the side chain of transition states and is normal for more associative reactions
Thr168 in an adjacent molecule and to Arg47 in the same mole- (35). Abolishment of general acid catalysis in PTPs is accompa-
cule. In the Michaelis complex structure, the respective aspartic nied with increase of the 15(V/K) KIE. The magnitude of 18(V/
acid hydrogen bonds to the backbone carbonyl of Gln166 in an K)bridge increases as well, due to the absence of the inverse
adjacent molecule. There are no other significant changes in contribution from protonation (see the entry for the D356A
the enzyme or peptide between Michaelis complex and TSA1 mutant of YopH) (36).
structures that can be attributed to crystal packing.
The experimental and most theoretical results indicate that
Fig. 3 combines the present TS structures with published the Michaelis complex consists of the dianion form of the sub-
ones to depict the active site arrangement at each step of the strate, with the nucleophilic cysteine in the thiolate form and
pathway. Supplemental Table S1 summarizes the structures the conserved Asp residue on the WPD-loop in the protonated
used for each step.
form, followed by a loose metaphosphate-like phosphoryl
transfer in which the leaving group is fully neutralized (2, 36,
59–62), although some theoretical studies have suggested that
PTPs utilize the monoanionic form of the substrate (63, 64).
DISCUSSION
Kinetic Isotope Effects and Transition State Structure
The KIEs for PTP1B are similar to those reported previously
for other PTPs (Table 2). Because they were measured using the
competitive method, the isotope effects are on the kinetic quan-
tity V/K, which includes events up to and including the first
irreversible step. These thus reflect the first catalytic step, phos-
phoryl transfer from substrate to enzyme.
Crystal Structures
An optimal transition state analog would consist of a peptide
sequence corresponding to a natural substrate incorporating a
tyrosine, with metavanadate (VO3) poised between the nucleo-
philic cysteine sulfur atom and the tyrosine hydroxyl group. In
solution, orthovanadate (VO34Ϫ) forms vanadate esters with
hydroxylic species, and these are found to be substrates for a
range of enzymes, including dehydrogenases, isomerases, and
aldolases (65). The formation constants of vanadate esters in
aqueous solution are on the order of 0.1–0.2 MϪ1 for aliphatic
esters and about 3 times larger for aromatic esters (65). One
precedent for vanadate ester formation and enzymatic recogni-
tion is the reaction between D-glucose and vanadate. D-Glucose
contains one primary and several secondary hydroxyl groups
that can form vanadate esters. Despite the many species that
form in solution, the small amount of glucose-6-vanadate that
forms is selectively recognized by glucose-6-phosphate dehy-
drogenase (66, 67). In the present work, the tyrosyl vanadate
ester of the peptide DADEYL was formed by preincubation of
Kinetic isotope effects reflect differences in bonding to the
labeled atom between the ground state and transition state (52).
Consequently, they are useful in probing transition state struc-
tures and reaction mechanisms of reactions. A primary kinetic
isotope effect at an atom undergoing bond cleavage will be nor-
mal (Ͼ1), due to the preference of the heavier isotope for the
lower energy (more tightly bonded) position. Secondary kinetic
isotope effects are normal when the labeled atom becomes
more loosely bonded in the transition state or inverse (Ͻ1)
when bonding becomes tighter. KIEs have been reported for a
5 Amino acid position numbers marked as positive and negative (AspϪ2
Leuϩ1, etc.) represent positions on the peptide relative to Tyr(P).
,
15878 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 21•MAY 21, 2010

The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
FIGURE 4. Coordination shell of the water W1 in the active site of PTP1B. a, first transition state; b, second transition state. Stereo images of a and b are
included in supplemental Fig. S5.
orthovanadate and peptide and was subsequently bound by substrate, it is difficult to say that the water molecule W1 is
PTP1B and co-crystallized.
involved in any proton transfer during catalysis. However, this
Vanadate anions form stable tetrahedral structures in solu- water molecule may be an important structural component of
tion but exhibit clear preference for trigonal bipyramidal struc- the active site through hydrogen bonding with key side chains.
tures in the presence of polydentate ligands (65), including the Additionally, this water may be an energy sink by partially sol-
active site of enzymes, such as PTPs (20, 22). In these enzymes, vating the phosphate moiety in the active site. It is important to
vanadate exhibits Ki values ranging from 0.4 to 5 ␮M (23, 68, 69) point out that Gln262 and Gln266, both in the coordination shell
and structural characteristics resembling the trigonal bipyra- of water W1, are highly conserved residues in PTPs.
midal transition state in phosphoryl transfer reactions. This
Each crystal structure depicted in Fig. 3 represents a discrete
geometry is formed by nucleophilic attack of the cysteine form of PTP1B in the ping-pong mechanism. Next, we provide
(Cys215 in PTP1B) on the vanadium atom, with the nucleophilic a step-by-step description of structural changes associated with
sulfur and leaving group oxygen occupying apical positions. binding and catalysis.
The equatorial oxygen atoms of this structure extensively inter-
Changes Accompanying Conversion of the Apoenzyme (PDB
Entry 2CM2) to the Michaelis Complex (MC) (PDB Entry 1PTU)
act with the P-loop (Fig. 2). The binding mode of vanadate in
the active site of PTPs has been considered a fair structural
transition state analog, showing clear advantages in relation to
other anions (24). For example, tetrahedral anions that do not
undergo similar stabilization in the active site of PTPs, exhibit
weaker inhibition. The Ki values for phosphate and arsenate
range from 10 to 30 mM (62, 70), and Ki values for tungstate
range from 5 to 60 ␮M (70, 71). It has been noted that in some
instances, PTP complexes with vanadate resemble a more asso-
ciative structure than the dissociative TS determined experi-
mentally (68).
P-loop—Substrate binding to the P-loop occurs with side
chain rotation of Arg221, which is rotated by Ϫ71° around ␹-3.
New hydrogen bonds are formed with the phosphate moiety of
the substrate, and with the carbonyl oxygen of Trp179. This
interaction stabilizes the WPD-loop in the active, closed
conformation.
WPD-loop—The closed position of this loop brings the gen-
eral acid Asp181 into position for catalysis (19, 46). Stabilization
of the closed conformation is further achieved by interaction of
the Phe182 side chain with the phosphotyrosine ring of the sub-
strate. Phe182 and the highly conserved Tyr46 flank the phenyl
ring of the substrate.
Information from the Crystal Structures
Binding of Substrate and Water Molecules in the Active Site
Substrate binding to PTPs is accompanied by the expulsion
Q-loop—The Gln262 residue occupies the same position in
of several water molecules from the active site. Three water both structures. Steric constraints in the MC prevent the Gln262
molecules are commonly found in crystal structures of apo- side chain from interactions with the active site, precluding a
PTPs at positions occupied by the phosphoryl nonbridge oxy- catalytic function in the first catalytic step. The side chain is in
gens when substrate is bound (31). Displacement of these sufficient proximity to participate in van der Waals interactions
waters may supply the entropic driving force assisting the sub- with the Tyr(P) side chain of the substrate.
strate binding. Other water molecules have important roles in
Lys-loop—Lys120 is rotated Ϫ100° around the ␹-4 angle. In
catalysis by PTP1B (29). The water molecule W1 in Fig. 4 is the apo structure, the ammonium moiety is embedded in a
found in all x-ray structures in the catalytic pathway of PTP1B polar environment interacting with the hydroxyl groups of
and is also observed in other members of the PTP family (e.g. Ser118, Ser216, and Tyr46. In the MC, the Lys120 side chain
Yersinia PTP (72) and PTP␤ (22)). In TSA1, the coordination hydrogen-bonds to the Asp181 and Glu115 side chains.
shell for W1 includes the side chains of Asp181 and Gln266, the
Tyr(P) Recognition Loop—The position of Asp48 is similar for
backbone amide proton of Phe182 in the WPD-loop, and axial the apo and MC states, but in the MC, the Asp48 side chain
and equatorial oxygens of the trigonal bipyramid structure. In hydrogen-bonds to the peptide main chain amides of Tyr(P)
TSA2, the coordination shell of W1 also includes the side chain and Leuϩ1. Peptide binding has a strong influence in the posi-
of Gln262. In view of the short distance between the side chain of tion of Arg47, which moves to make hydrogen bonds with Glu
Ϫ1
general acid catalyst Asp181 and the oxygen leaving group in the and the main chain carbonyl of Asp
.
Ϫ4
MAY 21, 2010•VOLUME 285•NUMBER 21
JOURNAL OF BIOLOGICAL CHEMISTRY 15879

The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
imity with the active site by steric
clashes with the vanadyl Tyr. This
verifies the proposal of Pannifer et
al. (29) that Gln262 has no catalytic
function in the first step.
Lys-loop—The Lys120 residue
rotates around the ␹-4 dihedral by
Ϫ20°. In both states, this residue
hydrogen-bonds to Asp181 and
Glu115. However, the interaction in
the TSA1 seems to be more favor-
able than in the MC; donor-accep-
tor distances involving Lys120 are
2.6 Å with Asp181 and 2.7 Å with
Glu115 but 0.2–0.3 Å longer in the
MC (PDB entry 1PTU). In the other
Michaelis complex (PDB entry
FIGURE 5. Close-up of details from crystal structures along the reaction pathway catalyzed by PTP1B.
a, initialstate, apoenzyme;b, Michaeliscomplex;c, firsttransitionstate;d, phosphoenzyme;e, secondtransition
state; f, product complex. The figure was prepared from superimposed structures, which are presented side by
side. In the first step, the phosphorus atom swings from the substrate to the nucleophilic cysteine, whereas the
positions of the leaving group oxygen, the nucleophile, and nonbridging oxygens do not change appreciably.
1G1G), Lys120 is found in a similar position as PDB entry 1PTU
and does not exhibit hydrogen bonds as short as those observed
in TSA1.
Changes Accompanying Conversion of the MC (PDB Entry
1PTU) to the Transition State Analog of the First Step, TSA1
(PDB Entry 3I7Z)
The TSA1 structure depicts a pentacoordinate species con-
Tyr(P) Recognition Loop—As in other PTP1B structures with
sisting of a planar metavanadate with apical ligands and equa- boundpeptides(PDBentries1EEO(75), 1G1G, and1G1H(46)), in
torial oxygen atoms interacting with the P-loop. This structure TSA1, Asp48 forms hydrogen bonds to the main chain amides of
ϩ1
models the TS of the first catalytic step.
vanadyl-Tyr(P) and Leu residues. The Asp48 residue is con-
P-loop—The Cys215 sulfur attacks the vanadyl center in line served among all PTPs and may be important in determining the
with leaving group departure. The nonbridge oxygen positions overall substrate orientation, in positioning the peptide substrate
are not appreciably different from their position in the MC, but for an optimal orientation for the attack of Cys215 and proton
the vanadium position has moved relative to that of phospho- transfer from Asp181 (46). Our TSA1 structure indicates that this
rus. The unusually low pKa of 4.6 for the Cys215 thiol (73, 74) interactionismaintainedinthetransitionstate. TheC␣positionof
results in part from a stabilizing hydrogen bond with Ser222
.
Asp48 movesby0.5ÅfromtheMCtotheTSA1structuresinorder
The sulfur-oxygen distance establishing this interaction is not to maintain proper structural requirements for the hydrogen bond
significantly different in the apo (3.2 Å), MC (3.0 Å) (this is between Asp48 and the peptide.
shorter because PDB entry 1PTU is a C215S mutant), and TSA1
The position of Arg47 differs from the MC 1PTU by about 3.5
(3.3 Å) structures. The constancy of this distance indicates that Å in the distal part of the side chain. This is probably related to
the low pKa of the nucleophile is permanently built into the the bond lengthening of the scissile bond observed from the
enzymatic structure.
ground to the transition state (Fig. 5). In the MC, weak hydro-
WPD-loop—The Asp181 carboxyl group moves closer to the gen bonds are present between Arg47 and GluϪ1 and the main
Tyr(P) leaving group oxygen (2.6 Å) than in the MC structure chain carbonyl of AspϪ4 of the peptide. There are no apparent
(3.5 Å). This distance is 3.0 Å in a related MC structure (PDB hydrogen bonds involving the Arg47 in TSA1, although it is
Ϫ1
entry 1G1G) with the peptide sequence ETDYpYRKGGKLL embedded in a position with negative charges from Glu
,
Ϫ4
bound to an inactive C215A mutant (46). In the first catalytic AspϪ2, and Asp
.
step, the phosphotyrosine is attacked by the nucleophilic Cys215
The combined changes in the positions of Arg47 and Asp181
accompanied by proton transfer from Asp181 to the leaving from the ground to the transition state reflect the needs of catal-
group oxygen. KIE data confirm that the efficiency of leaving ysis. In the transition state, the protein structure at the WPD-
group protonation is such that the leaving group is fully neu- loop becomes tighter to facilitate proton transfer but loosens
tralized in the TS.
around Arg47 in order to permit leaving group departure.
Comparing TSA1 with MC structure (PDB entry 1PTU), the
phenyl ring of Phe182 is rotated Ϫ23° around the ␹-1 dihedral; in
MC (PDB entry 1G1G), this residue is rotated 15°. The Ϫ23°
rotation of Phe182 permits extensive interaction between the
Changes Accompanying Conversion of TSA1 (PDB Entry
3I7Z) to the Phosphoenzyme (PDB Entry 1A5Y)
P-loop—Cys215 is phosphorylated. No other significant
Phe182 and the vanadyl tyrosine aromatic rings. However, in the changes are observed in this region.
MC 1G1G, the side chain of Argϩ1 is very close to the Phe182
WPD-loop—This loop slides horizontally, and Asp181 is dis-
ring, and because of van der Waals or steric clashes, the Phe182 placed 1.2 Å relative to its position in TSA1.
assumes a different position compared with MC 1PTU. This
Q-loop—A comparison is not possible in this case because
variability in the details of peptide-enzyme interactions fits PDB entry 1A5Y is a Q262A mutant.
with the substrate flexibility of PTP1B.
Lys-loop—In the phosphoenzyme, the Lys120 side chain is not
Q-loop—The Gln262 position does not change appreciably involved in hydrogen bonds with other protein residues. In
from its position in the MC and remains excluded from prox- TSA1, this residue is involved in hydrogen bonds with Asp181
15880 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 21•MAY 21, 2010

The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
and Glu115. These interactions may be important for position-
Lys-loop—In contrast to TSA1 and TSA2, in the intermediate
ing Asp181 during catalysis because this residue in the phos- and the product complexes, Lys120 is positioned away from
phoenzyme is found 1.2 Å away from its catalytically competent Asp181 and Glu115 and does not hydrogen-bond to protein
position observed in TSA1.
residues.
Tyr(P) Recognition Loop—The interactions between residues
Tyr(P) Recognition Loop—Relative to TSA1, Asp48 and Arg47
move away from their positions involved in hydrogen bonds Arg47 and Asp48 in TSA2 are lost in the product complex.
with the peptide. This loop backbone is similar to its position in
other structures without bound peptide.
Complementing these specific step-by-step observations, we
also notice that several precedents from the modeling of enzy-
matic features in small molecules indicate that decreased
entropy and proximity have a profound effect in nucleophilic
attack and general acid catalysis. In both cases, these effects
have shown catalytic contributions of over 106-fold (76–78),
Changes Accompanying Conversion of the Phosphoenzyme
(PDB Entry 1A5Y) to the Transition State Analog of the Second
Step, TSA2 (PDB Entry 3I80)
P-loop—The side chains and backbone positions of TSA1, which, combined, may partially explain the catalytic profi-
the phosphoenzyme, and TSA2 are very similar. We note that ciency of enzymes. Upon formation of the Michaelis complex in
the hydrogen bonding distances between the Cys215 sulfur atom PTPs, WPD-loop closure and the resulting proximity of the
and the oxygen of Ser222 (3.3 and 3.2 Å respectively) remain Asp181 side chain to the leaving group oxygen cause the pKa of
unchanged from those of the apo, MC, and TSA1 structures and the leaving group tyrosine to drop below its acidity in water. It is
are essentially constant through the entire catalytic cycle. In the interesting to observe that lengthening of the scissile P–O bond
transition state of the second step, this interaction serves to facili- causes the peptide chain to slightly shift in the active site of
tate the leaving group propensity of the sulfur atom.
PTP1B in TSA1 relative to the MC structure. This change prob-
WPD-loop—This loop is found in a similar position in TSA1 ably occurs upon transformation of the Michaelis complex to
and TSA2. In the phosphoenzyme structure, the Asp181 residue the transition state because the apical bond in the trigonal bipy-
is slightly displaced 1.2 Å away from its position in TSA1 and ramidal transition state is longer than the respective bond in the
TSA2. In the second catalytic step, this residue is a general base ground state. However, the phosphotyrosine ring (and conse-
for the deprotonation of a nucleophilic water molecule.
quently the leaving group oxygen) and nucleophile (Cys215) do
Q-loop—Comparing TSA2 with TSA1, the Gln262 residue is not appreciably change their position (Fig. 5). The major part of
rotated by Ϫ120° around the ␹-2 angle. This brings the amide the reaction coordinate, implied by these structures, is move-
moiety into the active site, where it positions the nucleophilic ment of the phosphorus atom between the leaving group and
water molecule for phosphoenzyme hydrolysis. This structure the nucleophile.
explains the observation that the Q262A mutation hinders phos-
phoenzyme hydrolysis but not the first step of catalysis (29).
The proximity of the general acid in TSA1, brought into
the active site by the conformational change associated with
Lys-loop—In the phosphoenzyme intermediate, the Lys120 WPD-loop closure, implies that the electronic effects of pro-
side chain is not involved in hydrogen bonds with other protein ton transfer to the ester oxygen, rather than only structural
residues. In TSA2, as in TSA1, Lys120 is involved in hydrogen consequences, are important for the very small leaving group
bonds with Asp181 and Glu115
.
dependence in PTPs. Site-directed mutagenesis and Bro¨n-
As revealed by the TSA1 and TSA2 structures, the Lys120 sted relationships for YopH, another member of the PTP
may interact with key residues in the transition state. This con- family, show that abolishment of general acid catalysis by
trasts with molecular dynamics simulations of the ground state, Asp is accompanied by a change of the Bro¨nsted ␤lg from
which observed concurrent movements between the WPD- about Ϫ0.1 in the wild type to Ϫ1 in mutants (70). In the
loop and Lys-loop during WPD-loop closure (31). Together, transition state, the general acid catalyst Asp181 may drive
these observations imply that the movement of the Lys-loop the proton transfer causing the P–O bond lengthening. The
probably lags behind the WPD-loop closure in the early stages charge in the phosphate moiety changes from Ϫ2 in the sub-
of each catalytic step.
strate to approximately Ϫ1 in the transition state. Negatively
Tyr(P) Recognition Loop—In TSA2, the side chains of Arg47 charged nucleophiles are unlikely to react with phosphate
and Asp48 hydrogen bond with each other. The backbone of monoesters in the dianion form. However, this has been
this region is in a similar position in the phosphoenzyme and observed in cases where, as in the PTP1B reaction, proton
TSA2.
transfer is present or the phosphate moiety is neutral or
The distances between the two apical oxygen atoms are iden- monoanionic (78).
tical within the uncertainty limits of the structure in the two
transition state analogs (Fig. 5). This implies a similar transition
state for the two phosphoryl transfer reactions.
CONCLUSIONS
The close similarity of the apical O–O distances in TSA1 and
TSA2 support the notion that the transition states of the two
catalytic steps are very similar. KIE and linear free energy rela-
tionship data have suggested that the first TS is loose for the
Changes Accompanying Conversion of TSA2 (PDB Entry
3I80) to the Product Complex (PDB Entry 2HNP)
P-loop and WPD-loop—Both loops return to an open confor- related PTPs, YopH and Stp (36, 62, 79), as is the second TS in
mation, as observed in the apo enzyme.
Stp1 (80). Because PTPs exhibit very similar active sites, it is
Q-loop—Relative to TSA2, in the product complex, Gln262 likely that the same is true in the transition states of reactions
rotates back to the position observed in the apo form.
catalyzed by other PTP family members.
MAY 21, 2010•VOLUME 285•NUMBER 21
JOURNAL OF BIOLOGICAL CHEMISTRY 15881

The Mechanism of Catalysis by Protein-tyrosine Phosphatase 1B
22. Evdokimov, A. G., Pokross, M., Walter, R., Mekel, M., Cox, B., Li, C.,
Several general conclusions are revealed by the step-by-step
analysis. The leaving group propensity in the first catalytic step
is modulated by Asp181 through an interaction that is con-
trolled by motions of a protein loop. In contrast, the pKa of the
Cys251 thiol (the nucleophile in the first step, the leaving group
Bechard, R., Genbauffe, F., Andrews, R., Diven, C., Howard, B., Rastogi, V.,
Gray, J., Maier, M., and Peters, K. G. (2006) Acta Crystallogr. D 62,
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23. Zhang, M., Zhou, M., Van Etten, R. L., and Stauffacher, C. V. (1997) Bio-
chemistry 36, 15–23
in the second) is controlled by a hydrogen bond in a fixed struc- 24. Davies, D. R., and Hol, W. G. (2004) FEBS Lett. 577, 315–321
25. Baxter, N. J., Olguin, L. F., Golicnik, M., Feng, G., Hounslow, A. M., Ber-
tural element that is unchanged throughout the catalytic cycle.
mel, W., Blackburn, G. M., Hollfelder, F., Waltho, J. P., and Williams, N. H.
The unusually low pKa makes catalytic sense in the second step,
(2006) Proc. Natl. Acad. Sci. U.S.A. 103, 14732–14737
in which Cys215 is a leaving group. The first step would logically
26. Fauman, E. B., Yuvaniyama, C., Schubert, H. L., Stuckey, J. A., and Saper,
be assisted by a weakened hydrogen bond that would raise this
pKa, but the evidence from the structures is that the low pKa is
fixed. Lys120 hydrogen-bonds to key residues involved in catal-
ysis, but its main function may remain in structural and pro-
tein-protein interactions. This may be an important factor for
development of PTP inhibitors. The transition states of the two
catalytic steps are indicated to be essentially identical. A struc-
tural rationale for the role of Gln262 in the second catalytic step,
but not the first, is now evident.
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