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Crystal Structures of Substrate-and Sulfate-Bound Mouse Thymidylate Synthase

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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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Pteridines / Vol. 20 / Special issue
163
A. Dowierciał et al. : Crystal structures of substrate- and sulfate-bound mouse thymidylate synthase
Pteridines
Vol. 20, Special issue, pp. 163-167
Crystal Structures of Substrate- and Sulfate-Bound
Mouse Thymidylate Synthase
Anna Dowierciał1, Adam Jarmuła1, Wojciech Rypniewski2, Monika Sokołowska3, Tomasz Frączyk1,
Joanna Cieśla1, Wojciech Rode
1 Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warszawa, Poland
2 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
3 International Institute of Molecular and Cell Biology, Polish Academy of Sciences, Warszawa, Poland
Abstract
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.
Key words : Thymidylate synthase (EC 2.1.1.45)
thymidylate synthase, 3IHH, 3IHI
Introduction
Thymidylate synthase (TS) is ubiquitous
among species and is one of the most conserved
enzymes known so far. It catalyzes the
reductive methylation of 2’-deoxyuridine 5’-
monophosphate (dUMP) in the presence of the
folate cofactor N5, N10-methylenetetrahydrofolate
(mTHF) serving as an one carbon donor
and reductant, to form deoxythymidine
monophosphate (dTMP) and dihydrofolate. Since
the reaction is the only de novo source of dTMP
required for DNA synthesis, TS is an important
target in chemotherapy, as its inhibition leads to
apoptosis of dividing cells. Numerous derivatives
of either dUMP or mTHF have been examined as
TS inhibitors and potential anticancer drugs, and
several have been in clinical use for years.
TS is a dimer, composed of ~30-37 kDa
monomers. The first step of the reaction
catalyzed by TS is the nucleophilic addition of
the active site cysteine (residue 189 in mTS) to
the nucleotide pyrimidine C(6). The position
and orientation of dUMP binding are essential
at this stage of catalysis, the former secured
by anchoring the nucleotide 5’- phosphate via
several conservative hydrogen contacts from
both TS subunits, and the latter promoted by
the hydrogen bonding between the conserved
asparagine (Asn220 in mTS) and both O4 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 – mTHF.
Among many TS crystal structures deposited
in the Protein Data Bank (PDB), only those
representing human and rat TS [1-8] are of
§Correspondence : Dr. Wojciech Rode, T: +48-22-589-2477;
F: +48-22-822-5342; Email: w.rode@nencki.gov.pl
Pteridines / Vol. 20 / Special issue
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A. Dowierciał et al. : Crystal structures of substrate- and sulfate-bound mouse thymidylate synthase
metazoan origin. Unlike in other known TS
structures, 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 mTS, containing
Lys157 corresponding to Arg163 in hTS, has
been hypothesized unable to populate the inactive
conformer [2], learning the crystal structure of
the mouse enzyme was of interest.
Materials and Methods
Reagents
K/Na tartrate, PEG 3350, PEG 4000, Li2SO4,
dUMP were purchased from Sigma, dithiothreitol
(DTT) was from Carl Roth GmbH.
Overexpression of mouse thymidylate synthase
The mTS coding region was cloned into
pPIGDM4+stop vector, expressed as HisTag-free
protein in thymidylate synthase-decient TX61- (a
kind gift from Dr. W. S. Dallas) E. coli strain and
purified as previously described (9). Phosphatase
inhibitors (50 mM NaF, 5 mM Na-pyrophosphate,
0.2 mM EGTA, 0.2 mM EDTA and 2 mM Na3VO4)
were present in the purication buffers. TS activity
was measured either spectrophotometrically [10]
or with the use of the tritium release assay [9].
The final preparation was highly homogeneous,
as judged by SDS/PAGE analysis of samples
containing up to 40 µg protein. Its specic activity
(the tritium release assay) was 1.75 µmol min-1 mg
protein -1 at 37oC.
Crystallization and data collection
Purified protein was dialyzed against 5 mM
Tris HCl buffer, pH 7.5, 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
4ºC at the following conditions. For mTS alone,
equal volumes of the protein (15 mg/ml) and well
solutions were mixed and allowed to equilibrate
with 0.5 ml of the well solution, containing 0.1
M Tris HCl, pH 8.5, 0.2 M Li2SO4 and 25% (w/
v) PEG 4000. For mTS-dUMP complex, a drop
of the protein solution (25 mg/ml, containing 5.9
mM dUMP) was mixed with an equal volume of
the well solution and allowed to equilibrate with
0.5 ml of well solution, containing 0.2 M K Na
Tartrate and 20% PEG 3350. The crystals were
transferred for a few seconds to cryoprotectant
solution containing either mother liquor and 25%
butanodiol (mTS–dUMP) or 30% glicerol (mTS
alone), and ash-cooled in N2 vapors.
X-ray diffraction data were collected from
three single ash-frozen crystals at the Max-Lab
Lund University Synchrotron using X-rays with
wavelengths of 1,038 and 0,908 Å.
Data processing. Structure determination and
renement
Data were processed with Denzo and Scalepack
[12]. Both structures were determined by
molecular replacement carried out with the
CCP4 package [13], using the rat TS ternary
complex without ligands as the search model. The
crystal structures of mTS and mTS-dUMP were
determined at resolutions of 1,94Å and 1,7Å,
respectively. The correctness of the two structures
was evaluated using Sfcheck & Procheck from
the CCP4 suite. Some X-ray data and model
renement parameters are summarized in Table 1.
Results and Discussion
The structures, consisting of one and three
dimers per asymmetric part of the unit cell for
mTS and mTS-dUMP, respectively (Figure 1
and Figure 2), showed an overall similarity to
the corresponding structures of hTS and rTS,
as expected for proteins with highly conserved
primary structures. The RMSD values with
respect to the crystal structures of hTS and rTS
(both TS-dUMP-Tomudex complexes) are 0.49,
0.47, 0.35 and 0.50 Å for mTS vs. hTS, mTS-
dUMP vs. hTS, mTS vs. rTS and mTS-dUMP vs.
rTS, respectively.
The molecule of dUMP was bound in a manner
similar to that observed in the corresponding
Table 1 : Summary of the X-ray data and model renement
mTS mTS-dUMP
Resolution [Å] 1.94 1.7
Number of reections 66 011 201 240
All atoms 4 932 14 789
Space group C2 C2
R-value (%) 22.0 24.8
Rfree-value (%) 26.9 29.5
rms bond 0.022 0.019
rms angle 1.9 1.8
Beamline I911-2 I911-5
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A. Dowierciał et al. : Crystal structures of substrate- and sulfate-bound mouse thymidylate synthase
complexes of other mammalian and bacterial
TSs, with the ligand 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 one subunit and
Arg169’ and Arg170’ from the other subunit) and
a single serine (Ser210). The orientation of dUMP
was secured by H-bonds between the conserved
Asn220 and the O4 and N(3)-H moieties of the
pyrimidine ring. The active site in the mTS
structure held a single sulfate anion, bound at
nearly the same location as dUMP phosphate
moiety in the mTS-dUMP structure and
stabilized by H-bonds with the pair of arginine
residues (Arg170’ and Arg209) from the quartet
coordinating the phosphate in the other structure.
The hTS loop 181-197 can populate two major
conformations, active and inactive, related to each
other by a 180 º rotation. While in the former
conformation, catalytic cysteine 195 locates itself
in the active site, directed toward C(6) of the
pyrimidine ring of dUMP (with which it forms
the thiol adduct), in the latter Cys195 is more than
10 Å away from the foregoing location, directed
toward the dimer interface of the enzyme. The
equilibrium between both conformations has
been shown to depend on the presence of either
phosphate/sulfate ions, driving the equilibrium
toward the inactive conformation, or dUMP,
driving it toward the active conformation.
In the mTS-dUMP structure, loop 175-191,
equivalent to human loop 181-197, populates
the active conformation, with catalytic Cys189
located at a close, but non-covalent distance
from dUMP. Interestingly, in the mTS structure,
loop 175-191 populates the same (active)
conformation, although the active site does not
contain dUMP. Of note is that it does contain
sulfate anion.
In hTS, the inactive conformer is stabilized
by a pair of hydrogen bonds from Arg163 to the
carbonyls of Ala191 and Leu192. The presence
of lysine, instead of arginine, at the position
157 in mTS (equivalent to hTS 163) disallows
Figure 1 : Mouse thymidylate synthase (chain A) with
sulfate anions (SO4
2-) bound inside the active sites (chains
A and B). The protein is colored by secondary structure
(helices in red, sheets in yellow, loops in green).
Figure 2 : Crystal structure of mouse TS complexed with
the substrate, dUMP.
Six subunits are shown, with ligands (blue) present in the
active sites.
Figure 3 : Superimposition of the loops 175-191, from
mTS-dUMP (lime), containing catalytic Cys189 inside
the active site (active conformation), and 181-197, from
hTS (blue), with catalytic Cys195 outside the active site
(inactive conformation). The rest of the structure of mTS-
dUMP (except loop 175-191) is shown in green.
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A. Dowierciał et al. : Crystal structures of substrate- and sulfate-bound mouse thymidylate synthase
the occurrence of the corresponding hydrogen
bonding (with Ala185 and Leu186) due to a
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 (Figure 3). This observation supports
the hypothesis of Lovelace et al [2].
The structures of both mTS and mTS-dUMP,
liganded with sulfate and dUMP, respectively,
differ from unliganded hTS in the conformation
of the loop 41-47 (hTS 47-53) (Figure 4). In
the liganded systems, loop 41-47 partly covers
the entrance to the active site, placing the side
chain of Arg44 sticking out toward the sulfate/
dUMP phosphate. In the absence of ligands, no
strong electrostatic attraction exists between
double negatively charged sulfate/phosphate and
positively charged Arg50, resulting in loop 47-
53 uncovering the entrance and extending away
from the active site.
Acknowledgments
Supported by the Ministry of Science and
Higher Education (grant no. N301 3948 33).
References
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Figure 4. Superimposition of mTS-dUMP subunit A
(green) and human R163K TS form 1 subunit A (blue).
Different conformations of the corresponding loops 41-
47 (mTS) and 47-53 (hTS) in the ligand-bound mTS and
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... Recombinant mouse TS protein, overexpressed and purified as described previously [7], was dialyzed against 5 mM Tris HCl buffer, containing 5 mM DTT and cocrystallized with the ligands at 4 ∘ C in the hanging drop composed of equal volumes (3.2 L each) of the protein solution (0.9 mM mTS), containing 6 mM dUMP and 6 mM Raltitrexed, and well solutions, containing 0.1 M MES pH 5.5-5.8, 5 mM DTT, 15.5% PEG 5K MME, and 0.2 M (NH 4 ) 2 SO 4 . ...
... It has been shown that in E. coli TS Trp80 (equivalent to mouse Trp103) is responsible for the proper orientations of Leu143 (equivalent to mouse Leu186) and of the cofactor molecule and that in the closed conformation the side chain of Leu143 plays a role in sequestering the active site from solvent [18]. Such a barrier for water molecules, formed by Leu186, is observed in chain A, but not in chain C, with orientations of Leu186 and Trp103 in the latter resembling those found in the previously presented binary mTS-dUMP complex [7]. ...
... In another mutant, V3L, a sharp drop in the catalytic activity has been assigned to Leu3 acting as a stabilizer of the inactive conformation of loop 181-197, with the latter preventing substrate binding. Comparison of the mTS binary (PDB ID 4E5O) [7] and tertiary complex presented here points to possible influence of nonpolar residues in the N-terminus on residues located in the active site through a series of hydrophobic interactions, including those between Leu2 and Val4, and amino acids in the protein interior, in particular Ile261 and Leu263 ( Figure 6). The latter two and other residues located in the protein segment 240-270, with the latter undergoing a considerable shift upon binding of Raltitrexed (Figure 7), may in turn interact with amino acids located in the proximity of the active site. ...
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.
... Mouse TS (mTS) recombinant protein, overexpressed and purifi ed as previously described [9], was dialyzed against 5 mM Tris HCl buff er, pH 7.5, containing 5 mM DTT and then concentrated using Amicon Centricon centrifugal fi lter. Crystals were grown by the vapor diff usion method in hanging drops at approximately 4 ° C (crystal A)/7 ° C (crystals B and C) at the following conditions. ...
... For both subunits of the dimer structure of the mTS-N 4 -OH-dCMP binary complex (structure A), the electron density map is well defined and continuous for the entire molecules of ligand and throughout the protein chains, except for poorly defined loops Glu 42-Thr 47. The mTS-N 4 -OH-dCMP structure is very similar to that of an analogous mTS complex with the substrate, dUMP [9] ( Figure 3 is located at a H-bond distance from the uracil O4 atom of dUMP. In the mTS-N 4 -OH-dCMP structure this water molecule (lower green) appears to be displaced by approximately 2.3 Å , thus being able to participate in a modified H-bonding network between the N 4 -OH group of N 4 -OH-dCMP and the Table 1 Data collection and refinement statistics for mTS-N 4 -OH-dCMP (crystal A) and mTS-N 4 -OH-dCMP-DHF (crystals B and C) structures. ...
... Unlike with the mTS-N 4 -OH-dCMP structure, although, the active site in the mTS-N 4 -OH-dCMP-DHF structure is closed, the catalytic Cys 189 being covalently bound to the C(6) atom of N 4 -OH-dCMP, reflected by the continuous electron density and distance of 1.87 Å between the C(6) and Cys 189 sulfur. Similar to the ligand molecules in the mTS-dUMP [9] and mTS-N 4 -OH-dCMP binary complexes, as well as dUMP molecule in the mTS-dUMP-ZD1694 ternary complex (PDB ID: 4EB4) (A. Dowiercia ł et al., unpublished), the molecule of N 4 -OH-dCMP is anchored in the active site by several H-bonds to its phosphate moiety from a group of four arginines and single serine (not shown). ...
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To solve the inhibition mechanism of thymidylate synthase (TS) by N4-hydroxy-dCMP (N4-OH-dCMP), crystallographic studies were undertaken. Structures of three mouse TS (mTS) complexes with the inhibitor were solved, based on crystals formed by the enzyme protein in the presence of either only N4-OH-dCMP [crystal A, belonging to the space group C 1 2 1, with two monomers in asymmetric unit (ASU), measured to 1.75 Å resolution] or both N 4-OH-dCMP and N5,10-methylenetetrahydrofolate (mTHF) (crystals B and C, both belonging to the space group C 2 2 21, each with a single monomer in ASU, measured to resolution of 1.35 Å and 1.17 Å , respectively). Whereas crystal A-based structure revealed the mTS-N4-OH-dCMP binary complex, as expected, crystals B- and C-based structures showed the enzyme to be involved in a ternary complex with N4-OH-dCMP and noncovalently bound dihydrofolate (DHF), instead of expected mTHF, suggesting the inhibition to be a consequence of an abortive enzyme-catalyzed reaction, involving a transfer of the onecarbon group to a hitherto unknown site and oxidation of THF to DHF. Moreover, both C(5) and C(6) inhibitor atoms showed sp3 hybridization, suggesting C(5) reduction, with no apparent indication of C(5) proton release. In accordance with our previous results, in all subunits of these structures the inhibitor molecule was identified as the anti rotamer of imino tautomer, forming, similar to deoxyuridine monophosphate, two hydrogen bonds with a conservative asparagine (mouse Asn220) side chain.
... 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]). ...
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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.
... 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 Pteridines and Folates (June 7-12, 2009, Jeju, Korea). ...
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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.
... A promising way of solving such problems is virtual selection of an inhibitor, based on comparison of the 3D structures of pathogen and mammalian enzyme proteins, aimed at non-conservative protein fragments differing between enzymes from both groups [ 5 ]. To make such an approach possible, crystal structures were solved of T. spiralis and C. elegans binary TS-dUMP complexes and structural comparisons were made with the corresponding mouse TS-dUMP complex [ 6 ]. Unfortunately, a similar comparison with the human enzyme has been so far impossible, as an analogous structure of the non-mutant human TS-dUMP complex is not available in the Protein Data Bank. ...
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Crystal structures were solved of the binary complexes Trichinella spiralis and Caenorhabditis elegans thymidylate synthases with deoxyuridine monophosphate (dUMP), with crystals obtained by the vapor diffusion method in hanging drops. For the T. spiralis thymidylate synthase-dUMP complex, the diffraction data were collected at the BESSY Synchrotron to 1.9 Å resolution. The crystal belongs to the space group P1 with two dimers in the asymmetric unit (ASU). For the C. elegans TS-dUMP complex crystal, the diffraction data were collected at the BESSY Synchrotron to 2.48 Å resolution, and the crystal belongs to the space group P 32 2 1, with two monomers (one dimer) in the ASU. Structural comparisons were made of both structures and each of them with the corresponding mouse thymidylate synthase complex.
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The crystal structures of a deletion mutant of human thymidylate synthase (TS) and its ternary complex with dUMP and Tomudex have been determined at 2.0 A and 2.5 A resolution, respectively. The mutant TS, which lacks 23 residues near the amino terminus, is as active as the wild-type enzyme. The ternary complex is observed in the open conformation, similar to that of the free enzyme and to that of the ternary complex of rat TS with the same ligands. This is in contrast to Escherichia coli TS, where the ternary complex with Tomudex and dUMP is observed in the closed conformation. While the ligands interact with each other in identical fashion regardless of the enzyme conformation, they are displaced by about 1.0 A away from the catalytic cysteine in the open conformation. As a result, the covalent bond between the catalytic cysteine sulfhydryl and the base of dUMP, which is the first step in the reaction mechanism of TS and is observed in all ternary complexes of the E. coli enzyme, is not formed. This displacement results from differences in the interactions between Tomudex and the protein that are caused by differences in the environment of the glutamyl tail of the Tomudex molecule. Despite the absence of the closed conformation, Tomudex inhibits human TS ten-fold more strongly than E. coli TS. These results suggest that formation of a covalent bond between the catalytic cysteine and the substrate dUMP is not required for effective inhibition of human TS by cofactor analogs and could have implications for drug design by eliminating this as a condition for lead compounds.