Crystal structures of the apo form and a complex of human LMW-PTP with a phosphonic acid provide new evidence of a secondary site potentially related to the anchorage of natural substrates

Article abstract of DOI:10.1016/j.bmc.2015.06.017

Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of single-domain enzymes with molecular weight up to 18 kDa, expressed in different tissues and considered attractive pharmacological targets for cancer chemotherapy. Despite this, few LMW-PTP inhibitors have been described to date, and the structural information on LMW-PTP druggable binding sites is scarce. In this study, a small series of phosphonic acids were designed based on a new crystallographic structure of LMW-PTP complexed with benzylsulfonic acid, determined at 2.1 ?. In silico docking was used as a tool to interpret the structural and enzyme kinetics data, as well as to design new analogs. From the synthesized series, two compounds were found to act as competitive inhibitors, with inhibition constants of 0.124 and 0.047 mM. We also report the 2.4 ? structure of another complex in which LMW-PTP is bound to benzylphosphonic acid, and a structure of apo LMW-PTP determined at 2.3 ? resolution. Although no appreciable conformation changes were observed, in the latter structures, amino acid residues from an expression tag were found bound to a hydrophobic region at the protein surface. This regions is neighbored by positively charged residues, adjacent to the active site pocket, suggesting that this region might be not a mere artefact of crystal contacts but an indication of a possible anchoring region for the natural substrate - which is a phosphorylated protein.

Full text of DOI:10.1016/j.bmc.2015.06.017

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Bioorganic & Medicinal Chemistry 23 (2015) 4462–4471
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Crystal structures of the apo form and a complex of human LMW-PTP
with a phosphonic acid provide new evidence of a secondary site
potentially related to the anchorage of natural substrates q
Emanuella M. B. Fonseca a, , Daniela B. B. Trivella a,b,à, Valéria Scorsato a, Mariana P. Dias a,§
,
Natália L. Bazzo a, Kishore R. Mandapati a,b, Fábio L. de Oliveira a,§, Carmen V. Ferreira-Halder c,
Ronaldo A. Pilli b, Paulo C. M. L. Miranda b, Ricardo Aparicio a,
⇑
a Laboratory of Structural Biology and Crystallography, Institute of Chemistry, University of Campinas, CP 6154, 13083-970, Campinas, SP, Brazil
b Department of Organic Chemistry, Institute of Chemistry, University of Campinas, CP 6154, CEP 13083-970, Campinas, SP, Brazil
c Department of Biochemistry, Institute of Biology, University of Campinas, CEP 13083-862, Campinas, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Low molecular weight protein tyrosine phosphatases (LMW-PTP, EC 3.1.3.48) are a family of single-
domain enzymes with molecular weight up to 18 kDa, expressed in different tissues and considered
attractive pharmacological targets for cancer chemotherapy. Despite this, few LMW-PTP inhibitors have
been described to date, and the structural information on LMW-PTP druggable binding sites is scarce. In
this study, a small series of phosphonic acids were designed based on a new crystallographic structure of
LMW-PTP complexed with benzylsulfonic acid, determined at 2.1 Å. In silico docking was used as a tool to
interpret the structural and enzyme kinetics data, as well as to design new analogs. From the synthesized
series, two compounds were found to act as competitive inhibitors, with inhibition constants of 0.124 and
0.047 mM. We also report the 2.4 Å structure of another complex in which LMW-PTP is bound to
benzylphosphonic acid, and a structure of apo LMW-PTP determined at 2.3 Å resolution. Although no
appreciable conformation changes were observed, in the latter structures, amino acid residues from an
expression tag were found bound to a hydrophobic region at the protein surface. This regions is
neighbored by positively charged residues, adjacent to the active site pocket, suggesting that this region
might be not a mere artefact of crystal contacts but an indication of a possible anchoring region for the
natural substrate—which is a phosphorylated protein.
Received 17 April 2015
Revised 29 May 2015
Accepted 5 June 2015
Available online 14 June 2015
Keywords:
LMW-PTP
Phosphatase inhibitors
Crystal structure
Enzyme kinetics
Anticancer drugs
Structure-based drug design
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
phosphorylation occurs to a much smaller extent than thre-
onine/serine phosphorylation, but it plays a pivotal role in cellular
Protein phosphorylation and dephosphorylation are the main
post-translational modifications of proteins in eukaryotic cells.1
These modifications are catalyzed by protein kinases and phos-
phatases that modify serine, threonine or tyrosine residues on dif-
ferent proteins, receptors, transcription factors and binding
proteins, thus controlling their biological functions. Tyrosine
signaling processes.2–5 The cellular level of the tyrosine phospho-
rylation is regulated by the opposing activity of protein tyrosine
kinases (PTKs) and protein tyrosine phosphatases (PTPs).6
Therefore, these enzymes control fundamental physiological pro-
cesses such as cell growth and differentiation, cell cycle, metabo-
lism, cytoskeletal function, and immune response. Accordingly,
deregulated activity of PTPs and PTKs is involved in the develop-
ment of numerous inherited and acquired human diseases such
as neurological and cardiovascular disorders, infections, diabetes,
cancer, and autoimmunity.7–9
q
PDB ID codes: 4Z9A, 4Z9B and 4Z99.
⇑
Corresponding author.
E-mail address: aparicio@iqm.unicamp.br (R. Aparicio).
Present address: Department of Biochemistry, Institute of Biology, University of
Campinas, CEP 13083-862, Campinas, SP, Brazil.
Alonso and co-workers10 demonstrated that human genome
contains 107 genes encoding both experimentally verified PTPs
and proteins with a domain homologous to the catalytic domain
of these PTPs. Among these genes, 81 are predicted to be active
protein phosphatases. Based on the primary structure of the
à
Present address: National Center for Research in Energy and Materials, Brazilian
Biosciences National Laboratory, 13083-100, Campinas, SP, Brazil.
§
Present address: Institute of Chemistry, University of Campinas, CP 6154, CEP
13083-970, Campinas, SP, Brazil.
http://dx.doi.org/10.1016/j.bmc.2015.06.017
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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4463
catalytic domains and the amino acid used in the catalytic reaction,
PTPs are subdivided into four evolutionarily distinct classes: I, II, III
(cysteine-based PTPs) and IV (aspartate-based PTPs). Despite the
three-dimensional structures of the catalytic domains of the cys-
teine-based PTPs are strikingly similar, they possess different
topologies and their regulatory domains vary significantly.10
In spite of relatively limited sequence similarity, the most sig-
nificant feature of the protein tyrosine phosphatase (PTP) super-
family is the conservation of the signature motif CX5R, which
forms the phosphate-binding loop in the active site (known as
the P-loop). This structurally conserved arrangement ensures an
identical catalytic mechanism where the cysteine and the con-
served arginine residues at catalytic site remain in close proximity
to hold the phosphate group of the substrate in a position for
nucleophilic attack by the cysteine thiol nucleophile.11
Low molecular weight protein tyrosine phosphatases (LMW-
PTP, EC 3.1.3.48) are a family of enzymes expressed in different tis-
sues with molecular weight up to 18 kDa. They are single-domain
enzymes and have been identified in a wide variety of organisms
including rat, human, bovine, bacteria, yeast and plants.12–19 In
humans, Class II cysteine-based PTPs are represented by the mem-
bers of LMW-PTP family (also known as acid phosphatase locus 1,
ACP1), which are widely expressed with no particular tissue-
specific expression. Four different human LMW-PTP messenger
RNA, derived by alternative splicing of a single transcript, have
been characterized. Two of them correspond to the classical active
35), in which water molecules are observed in the active site
pocket and a molecule of 1-naphtylacetic acid (NLA) is bound to
a surface region of the protein.
In the present study, a series of compounds was characterized,
and three new crystal structures of the isoform A (IF1) of LMW-PTP
were determined. These structures comprise one apo LMW-PTP
structure and two complexes with small molecule—one with the
hydrolysis product of the protease inhibitor PMSF (phenylmethyl-
sulfonyl fluoride) and other in complex with the PMSF analogous
benzylphosphonic acid. In both cases, the structures revealed that
these compounds bind non-covalently to the LMW-PTP catalytic
site, in a very similar fashion as predicted for the natural pTyr sub-
strate, therefore, acting as pTyr mimetic. Further, both in the apo
structure and in the complex with benzylphosphonic acid, an
unexpected crystallographic site diverse from the known active
site is occupied by amino acid residues from the construct expres-
sion tag.
2. Results and discussion
2.1. Sulfonic and phosphonic acids as LMW-PTP inhibitors
In the course of the first attempts to crystallize LMW-PTP in our
laboratory, we obtained the crystal structure of the enzyme in
complex with the hydrolysis product of the protease inhibitor
PMSF present in the protein lysis buffer. In an aqueous environ-
ment, PMSF can be easily hydrolyzed and the fluorine replaced
by a hydroxyl group, yielding benzylsulfonic acid, from now on
referred to as PMS (Fig. 1, compound 1). An additional structure
was also available in the literature, in which the active site was
occupied by a molecule of 2-(N-morpholino)ethanesulfonic acid
(MES buffering agent; PDB ID 5PNT3). The presence of a sulfonic
acid moiety both in PMS and MES molecules, which can mimic,
at some extent, the phosphate group of natural substrates, gave
us an initial clue about LMW-PTP and prompted us to design the
inhibitors based on phosphonic acids shown in Figure 1, com-
pounds 2 to 7.
isoforms
1 (IF1, PTPfast/isoform F or HCPTPA) and 2 (IF2,
PTPslow/isoform S or HCPTPB).20 Both isoforms are single polypep-
tide chains of equal length which display difference only in a short
sequence segment that corresponds to amino acid residues 40–73
in the mature protein. However, these isoforms present divergence
in their physical chemistry properties, especially with respect to
kinetics and consequently physiological functions.20–23
In recent years, PTPs have gained considerable attention as
important drug targets.24 However, despite potential inhibitors
have been designed, there are challenges in developing successful
LMW-PTP inhibitors. Firstly, the low bioavailability and it is very
common the observation of reactive oxygen species (ROS) produc-
tion by reported PTPase inhibitors, with a consequent PTP inhibi-
tion occurring by indirect and nonspecific ways.4,6,10,12,25–27 Since
LMW-PTP is proposed as a pharmacological target for cancer
chemotherapy,9,28 it is important to better understand the mecha-
nism and mode of binding of its inhibitors.
LMW-PTP (wild type or mutated) crystallographic structures of
a wide range of organisms were reported, most of them presenting
ions or other chemical substances contained in the sample buffer,
or the synthetic substrate p-nitrophenyl phosphate (pNPP).3,13,29–34
In the case of isoform A human enzyme, structural data are scarce,
with two crystal structures of isoform A reported to date, one in
which a molecule of 2-(N-morpholino)ethanesulfonic acid (MES
buffering agent) was observed in the active site (PDB ID 5PNT3)
and another deposited under the PDB ID 3N8I (unpublished Ref.
Phosphonic acids are a good choice for the design of phos-
phatase inhibitors since the replacement of the phenolic oxygen-
phosphorus bond in the natural substrate by a non-hydrolysable
carbon-phosphorus bond in the phosphonic acid do not alter sig-
nificantly the geometry or the electric charge distribution, afford-
ing appreciable inhibitory activities against these enzymes.36–38
Benzylphosphonic acid (Fig. 1, compound 2), in particular, was pre-
viously described as a weak inhibitor of phosphatases.36,37,39–41
Zhang and Van Etten reported that it is a competitive inhibitor of
bovine heart acid phosphatase, presenting an apparent inhibition
constant of 4.6 mM.42 Also, it inhibits a human placental alkaline
phosphatase (PLAP) and a bovine intestinal 50-nucleotide phospho-
diesterase with inhibition constants of 0.58 mM36 and 1.4 mM43
,
respectively. Benzylphosphonic acid has also been reported as an
inhibitor of Yersinia protein tyrosine phosphatase (YopH) and
Figure 1. Chemical structure of benzylsulfonic acid (PMS; 1) and the series of small molecules 2–7 designed and synthesized to probe phosphatase inhibitor recognition:
benzylphosphonic acid (2), 4-nitrobenzylphosphonic acid (3), 4-(chloromethyl)benzylphosphonic acid (4), diethyl 4-(chloromethyl)benzylphosphonate (5), diethyl
benzylphosphonate (6), diethyl (4-nitrobenzyl)phosphonate (7).

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E. M. B. Fonseca et al. / Bioorg. Med. Chem. 23 (2015) 4462–4471
PTP1B, with 5 mM and 3 mM as inhibition constants, respec-
tively.44 In fact, Stebbins et al. proposed that it can be a potential
pTyr mimetic bound to the catalytic site of YopH.45 To our knowl-
edge, this is the first study considering compound 2 as a human
LMW-PTP inhibitor.
an inhibitory constant (Ki) of 0.124 and 0.047 mM, respectively
(Fig. 2B and C, respectively).
When comparing 2, 3 and 4, it is reasonable to assume that the
substitution at the para position led to more effective ligands,
probably by favorable interactions at the edge of the active site.
Compounds 1 and 2, despite having almost the same shape, pre-
sented different inhibitions toward LMW-PTP although they
showed the very same position at the active site of the protein
on the complex crystal. It could be a result from the slight differ-
ence among conditions used in inhibition assays (pH 5) and crys-
tallization conditions (pH 6.5). Compound 1, which contains
sulfur, is approximately 1000 times more acidic than compound
2 and also has only one acid proton (pKa ꢀ0.6 for compound 1,
compared to 2.4 for the first proton and 7.8 for the second proton
of compound 2). So, compound 1 is expected to be totally dissoci-
ated in any of these pH and always bearing charge ꢀ1. Conversely,
compound 2 is expected to be 100% monodissociated at pH 5, but
50% monodissociated and 50% fully dissociated at crystallization
pH and may have charge ꢀ1 or ꢀ2 depending on pH value.46
Thus, considering that P-loop is composed of protonated and pos-
itively charged Arg18 and also several N–H peptidic bonds oriented
toward to center of the active site (residues Cys12 to Arg18), it is
expected that better hydrogen bond acceptors could interact with
this environment. Since sulfonate moiety has no bonded hydrogen,
it is able to accept a higher number of acidic hydrogen bonds from
the active site (P-loop). On the other hand, monodissociated phos-
phonic acid has only two free oxygen atoms and has a weaker
interaction with the active site. According to this rationale, com-
pound 1 would be more effective in inhibiting LMW-PTP, in agree-
ment with the results shown in Fig. 2A.
2.2. Enzyme kinetics and docking studies
The influence of the synthesized compounds on LMW-PTP
activity was evaluated by using the classical phosphatases sub-
strate p-nitrophenyl phosphate (pNPP) initially in a single concen-
tration activity assay. At a concentration of 2 mM, compounds 3
and 4 were more potent in inhibiting LMW-PTP (Fig. 2A). We thus
examined the inhibition mechanism of both compounds to evalu-
ate whether they were indeed acting as
a pTyr mimetic.
Compounds 3 and 4 act as competitive inhibitors and displayed
In order to provide a structural interpretation for the biological
assays, molecular modeling studies were done by using an exhaus-
tive rigid-body docking procedure. Our results indicated that com-
pounds 3 and 4 are able to mimic the interaction of the substrate
with the catalytic loop. The benzyl group establishes van der
Waals interactions with residues Glu50, Asp129 and Tyr131.
Figure S1A and B show a two-dimensional representation of the
mode of interaction of the compounds with LMW-PTP. Key interac-
tions responsible for the presence of the ligand in that position
include hydrogen bonds with amino acid residues Leu13, Gly14,
Asn15, Ile16, Cys17, and Arg18. Besides, compound 4 is a benzyl
chloride, which is very reactive and can undergo nucleophilic sub-
stitution reactions with hydroxyl, thiol and amine present in the
side chain of the protein.
Compounds 5, 6 and 7, on the other hand, have no shape com-
plementarity with the active site, since the ethyl group forming
esters are very bulky compared to the phosphate moiety.
Moreover, these small alkyl substituents decrease the possibility
of forming hydrogen bond or ionic interactions with the P-loop
residues of LMW-PTP in the bottom of the active site. According
to our docking results, these ester compounds are accommodated
in the opposite direction when compared to 1 or 2, as illustrated
in Figure S2. In addition, van der Waals interactions in the adjacent
region (residues Tyr49, Tyr132) of the active site were observed
with the ester groups.
2.3. New crystal structures of LMW-PTP
Human LMW-PTP consists of a four-stranded central parallel b-
sheet with flanking
a-helices on both sides. The active site consists
of a loop located between b-1 and
a-1 (Fig. S4), which contains the
consensus sequence motif CX5R (CLGNICR in the present case). The
complete amino acid sequence of the construct used in this study is
shown in Fig. S3. The protein construction included the His-tag
sequence MGSSHHHHHHSSGLVPRGSHMEF. The His-tag cleavage
by thrombin occurs between the residues arginine and glycine
Figure 2. Inhibition assays. (A) Single dose compound screening of phosphatase
inhibitors—compound concentration used was 2 mM; (B) Dixon plot for compound
3; (C) Dixon plot for compound 4. Compounds 3 and 4 act as competitive inhibitors
and displayed an inhibitory constant (Ki) of 0.124 and 0.047 mM, respectively.

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E. M. B. Fonseca et al. / Bioorg. Med. Chem. 23 (2015) 4462–4471
4465
(residues ꢀ7 and ꢀ6 marked in bold). For this reason, a remaining
polypeptide (residues ꢀ6 to ꢀ1, underlined), the expression tag,
remains bound to the protein N-terminal.
Arg18. Moreover, hydrogen bonds with N
e
and NH2 atoms from
Arg18 side chain were also observed. The benzyl ring is well
accommodated near Tyr131 side chain at the entrance of LMW-
PTP catalytic site (Fig. 3A), with van der Waals interactions occur-
ring with residues Cys12, Leu13, Ile16, Glu50, Asp129 and Tyr131.
A glycerol molecule, from cryoprotectant solution, was modeled
and is stabilized by hydrogen bond interactions with Glu93 and
Arg97. Interestingly, among the three structures, only in the PMS
complex, the side chain of Arg97 adopts a different conformation.
The same conformation was previously observed in the structure
reported as PDB ID 5PNT3 although without any apparent interac-
tion which would explain it. On the other hand, the conformation
adopted by Arg97 in the other two structures (apo LMW-PTP and
LMW-PTP:2) was already observed in PDB ID 3N8I,35 and can be
explained by crystal contacts with neighboring molecules (Fig. 3).
In the case of LMW-PTP in complex with 2, a total of 157 amino
acid residues could be modeled. In this experiment, crystals were
produced from protein expressed and purified in the absence of
PMSF to prevent an eventual interaction between the enzyme
and its hydrolysis product (PMS), as stated in Section 4.2, thus
allowing us to confirm compound 2 as the unique ligand candidate
to be present in solution. In the final structure, a non-contiguous
N-terminal was built, starting from the amino acid His-4 which
belongs to the expression tag included in the construct (Fig. S3).
No electron density was visible for residues Met0 to Ala4. In spite
of its negligible inhibitory activity, concentration of 2 in the crys-
tallization media was high enough to provide full occupancy of
the ligand atoms, which were found at the active site pocket
(Fig. 3B). Compound 2 exhibited virtually the same mode of bind-
ing observed in the complex with PMS. In summary, the oxygen
atoms of the phosphonate group were located at the bottom of
the active site, sitting on the phosphate binding pocket and form-
ing multiple hydrogen bonds with the nitrogen atoms of the active
LMW-PTP crystals contain a single molecule in the asymmetric
unit (ASU), in a molecular arrangement in which the active site is
unreachable through solvent channels due to an occlusion result-
ing from protein–protein crystal-packing contacts. This feature
limited the use of soaking to obtain complexes once the access of
small molecules to the active site is restricted. In spite of numerous
attempts, we did not succeed in obtaining another crystal form
of LMW-PTP. Therefore, attempts to obtain crystal structures of
LMW-PTP in complex with the compounds 2–7 were done by
co-crystallization.
Data collection and refinement statistics for complexes with
1
and 2 (PDB ligand codes PMS and B85, respectively) in
addition to an apo structure, are summarized in Table 1. The
expression tag interacts with a neighbor molecule both in the
complex with 2 and in the apo LMW-PTP structure, suggesting
the existence of
a secondary binding site, as discussed in
further detail below.
The refined model of LMW-PTP in complex with 1 contains 154
amino acid residues. The N-terminal was flexible, and electron
density was observed starting from residue Ala4 (Fig. S3). PMS
resembles a phosphorylated tyrosine (pTyr), which is the natural
substrate of PTPases. Not surprisingly, as a pTyr mimetic, the
ligand is recognized by the PTPase catalytic motif CX5R. The oxygen
atoms of PMS establish several interactions with the main chain
nitrogen of the residues Leu13, Gly14, Asn15, Ile16, Cys17 and
Table 1
Data collection and refinement statisticsa
Structure
LMW-
LMW-PTP:2 apo LMW-
site loop backbone, and NH2 and Ne atoms from Arg18 side chain.
PTP:1
PTP
Moreover, the edge is lined by the hydrophobic residues Leu13,
Ile16 and Tyr131, which form a suitable interaction surface that
can accommodate the benzyl group, and may interact with the
bound ligand (Fig. 3B).
In the apo structure, reliable electron density maps were found
for all of the 164 amino acid residues, comprising the whole con-
struct of LMW-PTP, including the entire expression tag. Despite
PDB ID
4Z9A
4Z9B
4Z99
Data collection statistics
Space group
P212121
P212121
P212121
Solvent content (%)
50.0
47.6
50.0
Cell dimensions (Å):
a
b
c
32.75
55.23
97.80
32.09
54.29
97.47
32.67
54.16
100.05
the presence of compound
3 in the crystallization solution
Resolution range (Å)
Last resolution shell (Å)
Multiplicity
28.07–2.10 27.88–2.41 36.75–2.30
(Section 4.4), no ligand is observed at the active site, which is occu-
pied by water molecules linked together by a hydrogen bond net-
work within the active site (Fig. 3C). Interestingly, the oxygen
atoms belonging to three of these water molecules are found at
almost the same position as the oxygen atoms of the phosphonate
group from compound 2 in the structure LMW-PTP:2 (Fig. S5).
Except for the N-terminal, no significant conformational
changes are observed in the protein chain when comparing the
three structures (average RMSD for common residues less than
1 Å). Both ligands 1 and 2 are well-ordered and the atom positions
fully occupied. Their modes of binding are nearly identical, which
is not surprising in view of their structural similarity and volume
(Fig. 1). Moreover, no structural changes in the active site environ-
ment were found to explain the difference observed in their inhibi-
tion effect nor apparent conformational changes in this region are
observed when comparing the structures reported here to the
structures 5PNT and 3N8I available at the PDB. The structural sim-
ilarity reveals that the presence of these compounds, regardless
their nature, cannot induce appreciable conformational changes
in the protein structure. All of the structures (both previously
reported and those described in the present work) share a common
crystal packing, belonging to a same space group (P212121), with
similar solvent content (ꢁ50%), cell dimensions and a single mole-
cule in the crystal ASU. Thus, we can argue that the differences
2.21–2.10 2.40–2.45
2.30–2.35
8.57
4.0 (4.0)
11.84
(11.58)
8.5 (50.8) 14.55
(56.18)
(7.94)
22.62
(62.10)
7.43
Rsym (%)
<I/r
(I)>
9.2 (2.2)
16.34
(2.94)
(2.0)
Refinement statistics
No. of reflections in refinement
Completeness (%)
No. of reflections used for Rfree (5%) 796
9846
97.59
6641
99.26
325
7967
99.68
385
R-factor—all reflections (%)
R-factor—excluding test set (%)
R-free (%)
protein atoms
water molecules
18.21
21.81
21.41
30.10
1260
69
19.43
19.02
28.59
1294
122
17.80
23.47
1245
88
Ligand atoms
27
11
0
Average B-factor for protein atoms
41.76
34.90
25.11
(Å2)
RMS deviations from ideality:
Bond lengths (Å)
Bond angles (°)
0.014
1.665
0.013
1.566
0.013
1.490
Ramachandran analysis
Favoured regions (%)
Allowed regions (%)
Outlier regions (%)
96.0
4.0
0.0
96.0
4.0
0.0
98.0
2.0
0.0
a
Values in parentheses refer to the last resolution shell.