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Mouse thymidylate synthase does not show the inactive conformation, observed for the human enzyme

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Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.
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ORIGINAL RESEARCH
Mouse thymidylate synthase does not show the inactive
conformation, observed for the human enzyme
Anna Dowierciał
1
Adam Jarmuła
1
Piotr Wilk
1
Wojciech Rypniewski
2
Monika Kowalska
3
Tomasz Fra˛czyk
1
Joanna Cies
´la
1
Wojciech Rode
1
Received: 2 July 2016 / Accepted: 29 August 2016 / Published online: 9 September 2016
ÓThe Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Crystal structures of mouse thymidylate syn-
thase (mTS) in complexes with (1) sulfate anion, (2) 20-
deoxyuridine 50-monophosphate (dUMP) and (3) 5-fluoro-
dUMP (FdUMP) and N
5,10
-methylenetetrahydrofolate
(meTHF) have been determined and deposited in Protein
Data Bank under the accession codes 3IHI, 4E5O and
5FCT, respectively. The structures show a strong overall
similarity to the corresponding structures of rat and human
thymidylate synthases (rTS and hTS, respectively). Unlike
with hTS, whose unliganded and liganded forms assume
different conformations (‘‘inactive’’ and ‘‘active,’’ respec-
tively) in the loop 181–197, in each of the three mTS
structures, the loop 175–191, homologous to hTS loop
181–197, populates the active conformer, with catalytic
Cys 189 buried in the active site and directed toward C(6)
of the pyrimidine ring of dUMP/FdUMP, pointing to pro-
tein’s inability to adopt the inactive conformation. The
binary structures of either dUMP- or sulfate-bound mTS,
showing the enzyme with open active site and extended
C-terminus, differ from the structure of the mTS–5-
FdUMP–meTHF ternary complex, with the active site
closed and C-terminus folded inward, thus covering the
active site cleft. Another difference pertains to the con-
formation of the Arg44 side chain in the active site-flank-
ing loop 41–47, forming strong hydrogen bonds with the
dUMP/FdUMP phosphate moiety in each of the two
liganded mTS structures, but turning away from the active
site entrance and loosing the possibility of H-bonding with
sulfate in the sulfate-bound mTS structure.
Keywords Thymidylate synthase Mouse Crystal
structure Active conformation
Introduction
Thymidylate synthase (TS; EC 2.1.1.45), a target in
chemotherapy, catalyzes the conversion of deoxyuridine
monophosphate (dUMP) and R-N
5,10
-methylenetetrahy-
drofolate (meTHF) to deoxythymidine monophosphate and
dihydrofolate, via reductive methylation, in which meTHF
serves as both methylene group donor and reducing agent
[1,2]. As the sole de novo source of thymidylate synthesis
in eukaryotic cells, for over 50 years, the enzyme has been
a target in chemotherapy [3], with active forms of drugs
being dUMP or N
5,10
-meTHF analogues that inhibit the
enzyme [4,5]. Between dUMP analogues active in
chemotherapy, the most prominent is 5-fluoro-dUMP
(FdUMP), a strong thymidylate synthase inhibitor being
the active form of drugs used in chemotherapy, such as
5-fluorouracil, 5-fluoro-20-deoxyuridine and 5-fluorocy-
tosine [2].
The first step of the reaction catalyzed by TS is the
nucleophilic addition of the active site cysteine (residue 189
in mouse TS) to the pyrimidine C(6) of the nucleotide. Of
essential role at this stage of catalysis are the proper binding
position and orientation of dUMP. The first is secured by
Anna Dowierciał and Adam Jarmuła have contributed equally to the
present work.
&Wojciech Rode
w.rode@nencki.gov.pl
1
Laboratory of Comparative Enzymology, Department of
Biochemistry, Nencki Institute of Experimental Biology,
Polish Academy of Sciences, Warsaw, Poland
2
Institute of Bioorganic Chemistry, Polish Academy of
Sciences, Poznan, Poland
3
International Institute of Molecular and Cell Biology, Polish
Academy of Sciences, Warsaw, Poland
123
Struct Chem (2017) 28:667–674
DOI 10.1007/s11224-016-0840-8
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
anchoring the phosphate of dUMP via several conservative
hydrogen contacts from both TS subunits, while the latter by
the way of hydrogen bonding between the conserved
asparagine (Asn 220 in mTS) and the O
4
and N
3
–H moieties
of the pyrimidine ring. Properly bound dUMP provides the
binding surface for the cofactor, resulting in the formation of
the ternary complex TS–dUMP–meTHF [2]. Following
activation of dUMP by the nucleophilic attack at the
pyrimidine C(6), the negative charge of cysteine residue is
delocalized, probably toward the C(4)=O of dUMP, and the
corresponding enolate anion is formed (Fig. 1) with its C(5)
position believed to become strongly nucleophilic. The latter
facilitates a consecutive attack of the methylene residue, in
the iminium ion form, resulting from opening of the imida-
zolidine ring of the cofactor. This leads to formation of the
covalent intermediate, ternary complex of the enzyme with
substrate and cofactor. Next, the C(5) hydrogen dissociates,
as proton, from dUMP pyrimidine ring, with concomitant b-
elimination of tetrahydrofolate, still remaining bound in the
active center. The latter enables hydride transfer from the
pteridine C(6), resulting in reduction of the pyrimidine C(5)
methylene group to methyl one. The reaction is completed by
regeneration of the pyrimidine 5,6 double bond, with con-
comitant elimination to form dTMP and enzyme [1,2].
Structure determination of human TS complexes
revealed a unique feature. In contrast to structures from
other species, human TS loop 181–197 was observed to
populate two conformers, one of them, apparently inactive,
stabilized by a pair of hydrogen bonds from Arg163 to the
carbonyls of Ala191 and Leu192. As mouse TS protein
contains lysine residue at the position corresponding to
arginine residue in human TS (Lys157 in mTS vs. Arg163
in hTS), it has been hypothesized unable to populate the
inactive conformer [6]. In order to test that hypothesis,
learning the crystal structure of the mouse enzyme was of
interest.
An initial account of a part of the present results has
been published in a special issue of Pteridines [7], covering
materials of the 14th International Symposium on Pteridi-
nes and Folates (June 7–12, 2009, Jeju, Korea).
Materials and methods
Reagents
K/Na tartrate, PEG 3350, PEG 4000, Li
2
SO
4
, dUMP were
purchased from Sigma, dithiothreitol (DTT) was from Carl
Roth GmbH.
Recombinant mouse thymidylate synthase
This was overexpressed and purified as described previ-
ously [8,9]. The enzyme was judged to be near homoge-
neous by denaturing polyacrylamide slab gel
electrophoresis using a 40, 20 and 10 lg sample. Enzyme
activity was measured spectrophotometrically [10], but at
the last purification step, the assay measuring tritium
release from [5-
3
H]dUMP was used as previously descri-
bed [11]. The specific activity of purified enzyme was
found to be 1.75 lmol min
-1
mg
-1
for conversion of
dUMP to dTMP at 37 °C.
Separation of non-phosphorylated fraction
of purified recombinant thymidylate synthase
preparation
The enzyme preparation was separated into phosphorylated
and non-phosphorylated fractions as previously described
[12]. Only the non-phosphorylated fraction was used for
crystallization.
Fig. 1 Thymidylate synthase-catalyzed reaction
668 Struct Chem (2017) 28:667–674
123
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Crystallization and data collection
Purified protein was dialyzed against 5 mM Tris HCl, pH
7.5 buffer containing 5 mM DTT and then concentrated
using Amicon Centricon centrifugal filter. Crystals were
grown by the vapor diffusion method in the hanging drops
at 4 °C under following conditions. For TS, equal volumes
of protein solution at 15 mg/ml concentration and well
solution were mixed and allowed to equilibrate with 0.5 ml
of well solution, containing 0.1 M Tris HCl, pH 8.5, 0.2 M
Li
2
SO
4
and 25 % (w/v) PEG 4000. For TS–dUMP com-
plex, a drop of protein solution at 25 mg/ml concentration,
containing 5.9 mM dUMP, was mixed as before 1:1 with
well solution and allowed to equilibrate with 0.5 ml of well
solution containing 0.2 M K Na Tartrate and 20 % PEG
3350. For TS–dUMP–meTHF complex, equal volumes of
mTS solution (25 mg/ml), containing 5 mM FdUMP and
10 mM meTHF and well solution (0.1 M Tris HCl, pH 8.5,
0.1 M MgCl
2
and 19.5 % (w/v) PEG 4000) were mixed
and allowed to equilibrate with 0.5 ml of well solution.
Crystallization conditions are summarized in Table 1.
The crystals (Figs. 3,4) were transferred for a few
seconds to cryoprotectant solution containing mother
liquor and 25 % butanodiol and 30 % glicerol for mTS–
dUMP and mTS, respectively, and flash-cooled in N
2
vapors.
X-ray diffraction data were collected from three single
flash-frozen crystals either at the Max-Lab Lund University
Synchrotron (mTS and mTS–dUMP), using X-ray wave-
lengths of 1.038 and 0.908 A
˚, or at the BESSY Syn-
chrotron (mTS–FdUMP–meTHF), using X-ray wavelength
of 0.918 A
˚.
Data processing, structure determination
and refinement
Data were processed with Denzo and Scalepack [13]. Both
structures were determined by molecular replacement
carried out with the CCP4 package [14], using the rat TS
ternary complex without ligands as the search model. The
crystal structures of mTS, mTS–dUMP and mTS–FdUMP–
meTHF were determined at resolutions of 1.94, 1.70 and
1.55 A
˚, respectively. The correctness of the structures was
evaluated using Sfcheck and Procheck from the CCP4
suite. Some X-ray data and model refinement parameters
are summarized in Table 2.
Results and discussion
The structures, consisting of either one dimer (mTS and
mTS–FdUMP–meTHF) or three dimers (mTS–dUMP) per
an asymmetric part of the unit cell, showed an overall
similarity to the corresponding structures of human and rat
TSs (hTS and rTS, respectively). The CaRMSD values
between the liganded and unliganded mTS structures pre-
sented here, as well as between these structures and the
crystal structures of other ternary complexes with dUMP
and Tomudex, including those with hTS [15,16], rTS [17]
and mTS [18], are shown in Table 3. The superimposition
of the mTS, mTS–dUMP and mTS–FdUMP–meTHF
structures is shown in Fig. 2. The similarity of the corre-
sponding TS structures is hardly unexpected in view of
highly conserved primary structures of different TS pro-
teins, as reflected by mTS amino acid sequence being 90 %
identical with those of hTS [19] and rTS [20], and about
40–55 % identical with prokaryotic and phage TS
sequences [19]. With respect to the latter, a recent com-
parison of amino acid sequences between TS of the para-
sitic nematode Trichinella spiralis and 33 other TS
proteins, both eukaryotic and prokaryotic, showed
43–68 % identities and 56–79 % similarities [21].
The mTS–dUMP structure showed the enzyme in the
active, open-state conformation. The dUMP pyrimidine
ring C(6) and the catalytic Cys189 were separated by dis-
tances of 3.19, 3.22, 3.19, 3.17, 3.28 and 2.94 A
˚(subunits
A, B, C, D, E and F, respectively), precluding covalent
bondings. The substrate molecule was bound in a manner
similar to that observed in the corresponding complexes of
other mammalian and bacterial TSs, with the ligand
Table 1 Crystallization conditions used
System Concentrations of TS and ligand(s) in the protein solution Composition of the well solution
mTS mTS (15 mg/ml) 0.1 M Tris HCl, pH 8.5 0.2 M Li
2
SO
4
No ligands 25 % (w/v) PEG 4000
mTS–dUMP mTS (25 mg/ml) 0.2 MK Na Tartrate
5.9 mM dUMP 20 % PEG 3350
mTS–FdUMP–meTHF mTS (25 mg/ml) 0.1 M Tris–HCl, pH 8.5
FdUMP (5 mM) 0.1 M MgCl
2
meTHF (10 mM) 19.5 % PEG 4000
Struct Chem (2017) 28:667–674 669
123
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anchored in the active site by several H-bonds to its
phosphate moiety from four arginines from both subunits
of the enzyme dimer (Arg44 and Arg209 from the first
subunit and Arg1690and Arg1700from the other subunit)
and single serine (Ser210). The orientation of dUMP was
secured by H-bonds between the conserved Asn 220 and
the O4 and N(3)-H moieties of the pyrimidine ring.
In the mTS–FdUMP–meTHF structure, the enzyme was
found in the active, closed state conformation, with the
catalytic Cys189 located in the active site and extended
toward FdUMP. The distances between mTS Cys189 cS
and FdUMP pyrimidine C(6) in the two subunits (A and B)
were equal, each 1.82 A
˚, pointing to covalent bonding.
However, the distances between the FdUMP pyrimidine
C(5) and meTHF C(11) atoms were 2.28 A
˚(subunit A) and
1.55 A
˚(subunit B), indicating covalent bond between the
two ligands to have been formed only in the subunit B.
FdUMP was bound slightly shifted (*0.9 A
˚), compared to
the position of dUMP in the mTS–dUMP structure, but
preserved all the non-covalent interactions of the latter
molecule. The conformation of the meTHF molecule was
stabilized by favorable contacts with Ile102 and Asn106
from a long loop–helix–loop segment (100–127), flanking
the entrance to the active site cleft, Ala305 and Met306
Table 2 Data collection and refinement statistics for mTS, mTS–dUMP and mTS–FdUMP–meTHF structures
Lattice type mTS (3IHI) mTS–dUMP (4E5O) mTS–FdUMP–meTHF (5FCT)
Monoclinic Monoclinic Monoclinic
Space group C 1 2 1 C 1 2 1 P 1 21 1
Unit cell parameters a=158.2 A
˚b=87.96 A
˚
c=68.04 A
˚
a=160.35 A
˚b=88.54 A
˚
c=136.76 A
˚
a=54.224 A
˚b=83.302 A
˚
c=68.850 A
˚
a=90°b=97.43°c=90°a=90°b=95.99°c=90°a=90°b=105.43°c=90°
Resolution range [A
˚] 19.48–1.94 19.95–1.7 26.14–1.55
No. of unique reflections 69,498 207,806 85,416
Multiplicity 3.6 (3.4) 6.4 5.9 (4.0)
\I/r(I)[15 16.9 14.0 (2.4)
No. of reflections used in
refinement
67,377 205,349 81,162
Rfactor [%] 22.0 23.5 12.0
R
free
factor [%] 26.6 29.2 16.6
RMSD bond [A
˚] 0.026 0.022 0.01
RMSD angle [°] 1.953 1.956 1.415
Table 3 CaRMSD [A
˚] in the pairs of crystal structures of mouse,
human and rat TSs in ternary complexes with dUMP and Tomudex
(4EB4, 1I00/1HVY and 2TSR, respectively), and with FdUMP and
meTHF (5FCT), and binary complex of mouse TS with dUMP
(4E5O), as well as sulfate-bound mouse TS (3IHI)
Structures Chains
AB CD A C B D
4E5O versus 3IHI 0.77 0.64 0.88
5FCT versus 3IHI 0.71 0.79 0.61
5FCT versus 4E5O 1.10 1.08 1.13
5FCT versus 2TSR 0.79 0.89 0.69
5FCT versus 1I00* 0.99 1.01 0.98
5FCT versus 1HVY* 1.05 1.10 1.00
5FCT versus 4EB4_AB 0.44 0.42 0.45
5FCT versus 4EB4_CD 1.05 1.27 0.78
* Two ‘‘generic’ conformations observed in the structures of the
ternary complexes of human
TS: open (1I00) and closed (1HVY)
Fig. 2 Alignment of the three mTS structures: sulfate-bound protein
(blue), binary complex with dUMP (green) and ternary complex with
FdUMP and meTHF (red). Proteins are shown as solid ribbons,
ligands as sticks and crystal waters as dots (Color figure online)
670 Struct Chem (2017) 28:667–674
123
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from C-terminus, Leu215 from a short loop 213–216 and
Phe219 from central a-helix 217–234 (Fig. 3). It should be
added that inhibition of thymidylate synthase by FdUMP
involves time-dependent formation of a ternary covalently
bound complex of the enzyme with FdUMP and mTHF, in
a reaction similar to that involving dUMP (in fact FdUMP
mimicks a substrate better than dUMP [22,23]). However,
at this step, the reaction stops, as the C(5) fluorine fails to
dissociate (due to the strength of the C–F bond), as it
happens with C(5) hydrogen in dUMP. What results is a
slowly reversible enzyme inactivation. Studies on the
mechanism of thymidylate synthase inhibition by FdUMP
were a rich source of information about the catalytic
mechanism [1,2].
The active site in the mTS structure held a single sulfate
anion, bound at nearly the same location as the phosphate
moieties of dUMP and FdUMP in the mTS–dUMP and
mTS–FdUMP–meTHF structures, respectively, and stabi-
lized by H-bonds with the pair of arginine residues
(Arg1700and Arg209), belonging to the quartet coordi-
nating the nucleotide phosphate in each of the liganded
structures. The presence at the active site of the sulfate
anion, an isosteric analogue of phosphate anion, is not
surprising, as 0.1 M Li
2
SO
4
was present in the crystal-
lization medium.
The structures of sulfate-bound mTS, and of the dUMP–
mTS and the mTS–FdUMP–meTHF complexes, differ in
the open/closed state equilibrium, revealing either an open
active site and extended C-terminus (mTS–dUMP and, to a
lower degree due to a shorter C-terminus, mTS) or closed
active site, with C-terminus folded inward and covering the
active site entrance (mTS–FdUMP–meTHF) (Fig. 4). The
latter is the most striking difference between the overall
similar structures, as reflected in modest CaRMSD values
for dimers, as well as individual subunits (Table 3).
Another, smaller difference pertains to the conformation of
the loop 41–47, located at one of the regions flanking the
active site cleft. In the liganded systems, this loop pene-
trates the active site, with the side chain of Arg44
extending toward, and forming H-bonds with the phosphate
moiety of dUMP/FdUMP. In the absence of ligands (and
despite the presence of sulfate, placed similarly as the
phosphate moieties of dUMP/FdUMP), Arg44 turns away
from the active site cleft and ‘‘uncovers’’ the active site
entrance (Fig. 5).
The closed-conformation structure of the mTS–
FdUMP–meTHF complex strongly resembles a mixed, yet
basically closed conformation observed in the AB dimer of
the previously reported mTS–dUMP-Tomudex (4EB4)
crystal structure (cf. RMSD in Table 3). However,
although still moderate, the RMS differences with respect
to the more open CD dimer, and in particular to subunit C
of the 4EB4 structure, are distinctly more pronounced.
Those differences are related to the open/closed active site
equilibrium and include the positions of C-terminus
(RMSD of 2.8–5.5 A
˚) and two segments on the opposite
flanks of the active site entrance, loop 41–47 (RMSD of
2.4–3.3 A
˚) and loop-helix-loop segment 100–127 (RMSD
of 2.0–3.5 A
˚) (Fig. 6).
In human thymidylate synthase, loop 181–197 can
populate two major conformations, active and inactive,
Fig. 3 Non-bonded contacts stabilizing the conformation of the
meTHF molecule in the subunit B of the mTS–FdUMP–meTHF
complex structure. Two alternative conformations of the meTHF
molecule are shown as ball and sticks, and other residues/moieties as
sticks. Atom coloring is by element: gray for carbons, red for
oxygens, blue for nitrogens, gold for sulfur and green for fluorine.
Hydrogen atoms are omitted for clarity. The alternative conformers of
meTHF and Lys 302 are colored cyan. The methylene bridge between
FdUMP and meTHF is colored purple. The hydrophobic contacts and
hydrogen bonds between meTHF and protein residues are colored
shades of violet and green, respectively (Color figure online)
Fig. 4 Differences between the active site conformations observed in
the three superimposed mTS structures: sulfate-bound protein (open
conformation; blue), binary complex with dUMP (open conformation;
green) and ternary complex with FdUMP and meTHF (closed
conformation; red) (Color figure online)
Struct Chem (2017) 28:667–674 671
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related to each other by a 180°rotation [24,25]. While
catalytic Cys 195 in the former conformation is present in
the active site, directed toward dUMP pyrimidine ring C(6)
and forming with it a thiol adduct, in the latter it is located
more than 10 A
˚away and directed toward the enzyme
dimer interface (Fig. 2). The equilibrium between both
conformations has been shown to be shifted toward the
inactive conformation in the presence of phosphate or
sulfate ions, with the active conformation promoted in the
presence of dUMP [25].
In the mTS–dUMP and mTS–FdUMP–meTHF struc-
tures, loop 175–191, equivalent to human loop 181–197,
populates the active conformation, with catalytic Cys189
either located at a close, but non-covalent distance from
dUMP, or covalently bound to FdUMP. Interestingly, in
the mTS structure, loop 175–191 populates the same (ac-
tive) conformation, despite the absence of dUMP/FdUMP
in the active site, containing only bound sulfate anion. This
is in contrast to the sulfate-bound human TS structure
(1HW4; Phan et al., 2001b), where corresponding loop
181–197 occupies the inactive conformation (Fig. 7).
In the human TS, the inactive conformer is stabilized by
a pair of hydrogen bonds from Arg163 to the carbonyls of
Ala191 and Leu192 (Fig. 3). The presence of lysine,
instead of arginine, at the position 157 in mTS (equivalent
to hTS 163) disallows the occurrence of the corresponding
hydrogen bonding (with Ala185 and Leu186) due to shorter
side chain and lower adaptability to multiple H-bonding of
the former, compared to the latter residue, as concluded
from the comparison of the mTS and hTS crystal structures
(Fig. 8). This observation supports the above mentioned
hypothesis by Lovelace et al. [6].
In view of the foregoing conclusion of obvious interest
is potential functional consequence of this mechanism, as
well as possibility to find structural features enabling the
inactive conformation stabilization (arginine residue in a
position homologous to hTS 163) in other TS structures.
Fig. 5 Differences in the conformation of the side chain of Arg44
from AS-flanking loop 41-47. Structures are colored as follows:
mTS—blue, mTS–dUMP—green, mTS–FdUMP–meTHF—red. Pro-
teins are shown as solid ribbons and ligands as sticks. Hydrogen
bonds are shown as dashed lines (Color figure online)
Fig. 6 Superimposition of subunit A from the mTS–FdUMP–meTHF
structure (red) and subunit C from the mTS–dUMP-Tomudex
structure (yellow). Most significant differences can be observed in
the conformations of C-terminus as well as two AS-flanking regions:
41–47 and 100–127. Proteins are shown as solid ribbons and ligands
as sticks (Color figure online)
Fig. 7 Superimposition of the sulfate-bound structures of mTS (3IHI;
green) and hTS (1HW4; red). Shown are fragments containing loops
175–191 from mTS (lime), with catalytic Cys189 inside of the active
site (active conformation), and 181–197 from hTS (pink), with
catalytic Cys195 outside of the active site (inactive conformation).
Cys195 (hTS) and Cys189 (mTS), as well as the sulfate anions from
both structures, are shown as sticks, and the rest of structures as solid
ribbon (Color figure online)
672 Struct Chem (2017) 28:667–674
123
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Considering strong influences of posttranslational modifi-
cations on kinetics and inhibition of TS-catalyzed reaction
[9,26,27] and resulting wide variability of properties of
the enzyme preparations of the same specific origin
[2,9,27], a simple comparison of mTS and hTS properties
would not answer the former question. However, Gibson
et al. [28] obtained a mutant R163K of the human enzyme
and found its catalytic activity to be higher than that of the
parental hTS. Furthermore, the native mutant protein
crystallized in the active conformation. Thus, hTS Arg163
appears to result in certain conformational plasticity,
associated with somewhat different catalytic properties.
Based on analysis of so far determined mammalian TS
protein sequences, indicating arginine to be present at
position homologous to hTS Arg163 in all primates, except
of prosimians, Luo et al. [29] suggested this enzyme form
to evolve from a common ancestral mammalian form (with
homologous Lys) following the divergence of prosimians
and simians. In accord, recently published mammalian TS
sequences show Lys at position homologous to hTS
Arg163 (NCBI Accessions EHB12689, ELK31636,
EPY81457 and EGW14385), with one exception of mar-
moset (NCBI Accession JAB35844), a representative of
New World Primates, showing corresponding arginine. The
letter confirms that this mechanism of local conformational
plasticity may be unique for primate TSs, at least between
mammalian enzyme forms. It should be mentioned, though,
that it may be not unique between other TSs, as at least the
enzyme sequences corresponding to both bacterium
Bacillus subtilis b and fungus Cryptococcus neoformans
show arginine residue in a position homologous to hTS 163
[1].
Acknowledgments Supported by the Ministry of Science and Higher
Education (Grant No. N301 3948 33) and National Science Centre
(Grant No. 2011/01/B/NZ6/01781).
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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... In accord, N 4 -OH-dCMP remained bound to the enzyme under conditions of the SDS electrophoresis [15]. Of note is that in a ternary mTS complex, formed in the presence of FdUMP and meTHF (PDB ID: 5FCT; Table 1), all three components were covalently bound [13], as expected based on the corresponding TS structures published previously [12]. The latter proves meTHF to be stable under crystallization conditions, thus confirming that the previously mentioned structure (PDB ID: 4EZ8) is not an artifact. ...
... The lack of clearly defined electron density for the final few (four in this case) residues of the C-terminus of subunit B has been observed previously for several structures of TS complexes, e.g., rat TS-dUMP-Tomudex (PDB ID: 1RTS and 2TSR [17]), CeTS-dUMP-Tomudex (PDB ID: 5NOO [18]), and mTS-dUMP-Tomudex (PDB ID: 4EB4 [19]). Spatial orientation of His190 present in the 4EZ8 structure (or of the corresponding histidine residue in any other specific variant of TS) is also found in multiple other structures of the mouse enzyme, e.g., 4EIN (mTS-N 4 -OH-dCMP complex [14]), 4E5O (mTS-dUMP [20]), 6F6Z (mTS-N 4 -OH-dCMP soaked with meTHF), 4EB4 (mTS-dUMP-Tomudex [19]), 3IHI (mTS apoenzyme [13]), and as one of two alternative conformations in 5BY6 (TspTS-dUMP [18,21]). On the other hand, an alternative orientation of His190 present in the 5M4Z structure is also observed in several other structures, including 5BY6 (as the other alternative conformation with 0.6 occupancy), 4PSG (CeTS-N 4 -OH-dCMP complex), 5NOO (CeTS-dUMP-Tomudex [18]), 5FCT (mTS-FdUMP-meTHF [13]), 4IRR (CeTS-dUMP [19]), 4IQB (CeTS apoenzyme [18]), and 4ISW (phosphorylated CeTS-dUMP [22]). ...
... Spatial orientation of His190 present in the 4EZ8 structure (or of the corresponding histidine residue in any other specific variant of TS) is also found in multiple other structures of the mouse enzyme, e.g., 4EIN (mTS-N 4 -OH-dCMP complex [14]), 4E5O (mTS-dUMP [20]), 6F6Z (mTS-N 4 -OH-dCMP soaked with meTHF), 4EB4 (mTS-dUMP-Tomudex [19]), 3IHI (mTS apoenzyme [13]), and as one of two alternative conformations in 5BY6 (TspTS-dUMP [18,21]). On the other hand, an alternative orientation of His190 present in the 5M4Z structure is also observed in several other structures, including 5BY6 (as the other alternative conformation with 0.6 occupancy), 4PSG (CeTS-N 4 -OH-dCMP complex), 5NOO (CeTS-dUMP-Tomudex [18]), 5FCT (mTS-FdUMP-meTHF [13]), 4IRR (CeTS-dUMP [19]), 4IQB (CeTS apoenzyme [18]), and 4ISW (phosphorylated CeTS-dUMP [22]). ...
Article
Full-text available
Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.
... Analyses of crystal structures of mTS bound with N 4 -OH-dCMP suggest that the molecular mechanism of an apparent strong preference for the anti rotamer of the imino inhibitor form, described in [43], is connected with the His190 presence. It was confirmed by both crystallographic studies [16,44] and molecular dynamics simulations [27] that the presence of His190 causes a steric hindrance preventing the syn form from being bound (Figure 9). ...
... Electrostatic potential computations for the crystal structures of mouse thymidylate synthase alone (mTS, PDB ID: 3IHI) [44] and in binary complexes with dUMP (PDB ID: 4E5O) [44] and N 4 OH-dCMP (PDB ID: 4EIN) [16] have been performed with continuum electrostatic calculations using Adaptive Poisson-Boltzmann Solver (APBS) v. 1.5 [87,88]. First, the crystal structures were prepared for APBS computations with PDB2PQR v. 2.1.1 [89], enabling the estimation of titration states and adequate protonation of molecules and the generation of pqr files with atomic charges and radii. ...
... Electrostatic potential computations for the crystal structures of mouse thymidylate synthase alone (mTS, PDB ID: 3IHI) [44] and in binary complexes with dUMP (PDB ID: 4E5O) [44] and N 4 OH-dCMP (PDB ID: 4EIN) [16] have been performed with continuum electrostatic calculations using Adaptive Poisson-Boltzmann Solver (APBS) v. 1.5 [87,88]. First, the crystal structures were prepared for APBS computations with PDB2PQR v. 2.1.1 [89], enabling the estimation of titration states and adequate protonation of molecules and the generation of pqr files with atomic charges and radii. ...
Article
Full-text available
A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson–Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme–ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the ”abortive reaction” inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.
... It has been shown that the inactive conformation of human TS is stabilized due to the presence of double hydrogen bonding between the side chain of Arg163 and the carbonyl oxygens of Ala191 and Leu192 [43]. Replacement of arginine with lysine in mouse TS (Lys157 in place of Arg163) prevents formation of the inactive conformer due to structural conditions forestalling the hydrogen bonding with lysine [111]. Replacement of arginine with glutamate in CeTS (Glu165 in place of Arg163) preserves the effect of replacement with lysine and causes the nematode enzyme to populate the active conformation. ...
... The active site of each CeTS subunit contains a single sulfate anion, located similarly as the phosphate group of dUMP in the CeTS-dUMP and CeTS-dUMP-Raltitrexed complexes. The same was previously observed for the corresponding mouse (PDB ID: 3IHI [111]) and human (PDB ID: 4H1I [112]) enzyme structures. The sulfate anions in the CeTS structure are loosely bound by the protein, as indicated by their relatively high temperature factors (45 and 37 Å 2 in subunits A and B, respectively), distinctly exceeding the average isotropic temperature factor for the whole model (16.7 Å 2 ). ...
... In search of unique fragments distinguishing the nematode from mammalian TSs, a thorough comparison between the structures of TsTS-dUMP and CeTS-dUMP and the analogous structures of mTS-dUMP (PDB ID: 4E5O [111]) and hTS-dCMP (PDB ID: 5WRN; not published yet) was conducted. Note that the hTS-dCMP structure was accepted for comparison in view of the lack in PDB of the hTS-dUMP structure. ...
... A long F-Trp109 distance, from 5.8 Å to 6.1 Å, is also observed in the hTS:FdUMP binary complex (PDB: 6QXG [36]), in which the side chain of Trp109 is rotated by~43 • away from the inhibitor with respect to the orientation observed for the same residue in the ternary complex with RTX. On the other hand, the same short-distance interaction between the FdUMP fluorine atom and the Trp103 side chain is observed in the ternary complex of mouse TS with FdUMP and 5-methyl-tetrahydrofolic acid (PDB: 5FCT [37], Trp103 of mouse TS matches Trp109 of the human enzyme). Leaving out the Cys195 Sγ -dUMP-C6 covalent bond observed in the 1HVY structure [15], the RMSDs between FdUMP and dUMP poses within the hTS active site are within the experimental error (0.5-0.8 Å < 3σ on atomic positions) in all four structures of the ternary complexes. ...
... FdUMP is covalently bound to the catalytic cysteine in some ternary complexes of nonhuman TSs with THF or mTHF such as bacterial [39][40][41] and mouse source [37] Overall, these observations suggest that it is the presence of the reduced cofactor (mTHF) or an analog (THF), rather than of a folate-like inhibitor that favors formation of a covalent bond between dUMP and TSs. Therefore, it is reasonable that our ternary complex does not show a covalent link to the protein. ...
Article
Full-text available
Combining drugs represent an approach to efficiently prevent and overcome drug resistance and to reduce toxicity; yet it is a highly challenging task, particularly if combinations of inhibitors of the same enzyme target are considered. To show that crystallographic and inhibition kinetic information can provide indicators of cancer cell growth inhibition by combinations of two anti-human thymidylate synthase (hTS) drugs, we obtained the X-ray crystal structure of the hTS:raltitrexed:5-fluorodeoxyuridine monophosphate (FdUMP) complex. Its analysis showed a ternary complex with both molecules strongly bound inside the enzyme catalytic cavity. The synergistic inhibition of hTS and its mechanistic rationale were consistent with the structural analysis. When administered in combination to A2780 and A2780/CP ovarian cancer cells, the two drugs inhibited ovarian cancer cell growth additively/synergistically. Together, these results support the idea that X-ray crystallography can provide structural indicators for designing combinations of hTS (or any other target)-directed drugs to accelerate preclinical research for therapeutic application.
... The Sγ of the catalytic C170 residue was at a distance of about 3.3 Å from the C6 of dUMP uracil moiety. These contacts are conserved across other ternary TS structures [13,[18][19][20][33][34][35]. These interactions were further strengthened by the hydrophobic interactions between the dUMP and the pteridine ring of methotrexate, running parallel to each other. ...
Article
Full-text available
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures.
... However, the same activity observed with FdUMP points to its being rather a permanent feature of the enzyme. It is of note that the crystal structure (PDB ID: 5FCT) of mouse TS complex formed with FdUMP and meTHF does not show any unusual properties [26], as compared to other corresponding TS structures (attempts to crystallize a complex of TS with FdUMP and THF have been so far unsuccessful). Therefore it is of interest whether the enzyme could use THF to reduce any other compounds, with or without a resulting self-inactivation effect. ...
Article
In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused "uncoupling" of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.
... Dowierciał et al. [64] reported the synthesis and characterization, including X-ray single-crystal structure determination, of mouse thymidylate synthase in complexes with (a) sulfate anion, (b) 2′-deoxyuridine 5′-monophosphate, and (c) 5fluoro-2′-deoxyuridine 5′-monophosphate and N 5,10 -methylenetetrahydrofolate. The results suggest that compared with human thymidylate synthase, the mouse thymidylate synthase did not show the inactive conformation observed for the human enzyme. ...
Article
Full-text available
The contents of issues 3 and 4 of Structural Chemistry from the calendar year 2017 are summarized in the present review. A brief thermochemical commentary and recommendations for future research have been added to the summary of each paper.
... All radiographic 3D holo structures of selected targets (BCL-2, PBD ID: 4AQ3 [27]; EGF, PDB ID: 3W33 [28]; Enoyl-acp Reductase, PDB ID: 1QSG [29]; FAK, PDB ID: 4KAO [30]; GSK-3 beta, PDB ID: 1Q41 [31]; HDAC, PDB ID: 5IX0 [32]; MetAP, PDB ID: 1YW7 [33]; telomerase, PDB ID: 5CQG [34]; TS, PDB ID: 4E5O [35] and VEGF, PDB ID: 3VO3 [36]) were retrieved from the Brookhaven protein data bank (http://www.rcsb.org/) with resolutions in the range of 1.52e2.40 ...
Article
Aims 5-Fluorouracil (5-FU), a thymidylate synthase (TS) inhibitor, has been used as the first-line chemotherapeutic drug for cholangiocarcinoma (CCA). The side effects and drug resistance have developed the limits of the clinical application of 5-FU in CCA treatment. Upregulation of Forkhead box M1 (FOXM1) and TS were shown to play a significant role in 5-FU resistance. In this study, the effect of Siomycin A (SioA), a FOXM1 inhibitor, on enhancing 5-FU cytotoxicity and reversing 5-FU resistance in CCA cell lines were demonstrated. Main methods Human CCA cell lines, KKU-100 and KKU-213A were used. Cell viability was determined using MTT assay. Expression of FOXM1 and TS proteins were determined using Western blotting. FOXM1 mRNA expression was quantitated using real-time PCR. The combination and dose reduction (DRI) were analyzed according to the Chou and Talalay method. Key finding Single drug treatment of 5-FU and SioA effectively inhibited CCA cell growth in dose and time dependent fashions. The two CCA cell lines had different responses to 5-FU but exhibited similar sensitivity to SioA. FOXM1 and TS expression were increased in the 5-FU treated cells but were suppressed in the SioA treated cells. A direct binding of SioA, to TS and 5,10-methylene-tetrahydrofolate as an inactive ternary complex was simulated. The combined treatment of 5-FU with SioA showed a synergistic effect with a high DRI and restored 5-FU sensitivity in the 5-FU resistant cells. Significance Targeting FOXM1 using SioA in combination with 5-FU might be a strategy to overcome the 5-FU resistance in CCA.
Article
Full-text available
The crystal structure of mouse thymidylate synthase (mTS) in complex with substrate dUMP and antifolate inhibitor Raltitrexed is reported. The structure reveals, for the first time in the group of mammalian TS structures, a well-ordered segment of 13 N-terminal amino acids, whose ordered conformation is stabilized due to specific crystal packing. The structure consists of two homodimers, differing in conformation, one being more closed (dimer AB) and thus supporting tighter binding of ligands, the other more open (dimer CD) and thus allowing weaker binding of ligands. This difference indicates an asymmetrical effect of the binding of Raltitrexed to two independent mTS molecules. Conformational changes leading to a ligand-induced closing of the active site cleft are observed by comparing the crystal structures of mTS in three different states along the catalytic pathway: ligand-free, dUMP-bound and dUMP- and Raltitrexed-bound. Possible interaction routes between hydrophobic residues of the mTS protein N-terminal segment and the active site are also discussed.
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Crystal structures of mouse thymidylate synthase (mTS), liganded with either 2'-deoxyuridine 5'-monophosphate (dUMP) or the sulfate anion, have been determined and deposited in Protein Data Bank under the accession codes 3IHH and 3IHI, respectively. The structures show a strong overall similarity to the corresponding structures of rat (rTS) and human (hTS) thymidylate synthases. The loop 175-191, corresponding to the hTS loop 181-197, populates the active conformation, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP. Another loop, 41-47, differs in conformation from the corresponding loop 47-53 in unliganded human enzyme. It folds due to electrostatic attraction between Arg 44 and the sulfate/dUMP phosphate and partly covers the entrance to the active site.
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