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Crystal structures of thymidylate synthase from nematodes, Trichinella spiralis and Caenorhabditis elegans, as a potential template for species-specific drug design

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Abstract

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.
DOI 10.1515/pterid-2013-0011Pteridines 2013; 24(1): 87–91
Anna Dowiercia ł , Piotr Wilk , Wojciech Rypniewski , Tomasz Fr ą czyk , Adam Jarmu ł a ,
Katarzyna  Banaszak , Magdalena D ą browska , Joanna Cie ś la and Wojciech Rode*
Crystal structures of thymidylate synthase
from nematodes, Trichinella spiralis and
Caenorhabditis elegans , as a potential
templatefor species-specific drug design
Abstract: Crystal structures were solved of the binary com-
plexes Trichinella spiralis and Caenorhabditis elegans thy-
midylate 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 col-
lected 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 cor-
responding mouse thymidylate synthase complex.
Keywords: 3D structure; nematode; thymidylate synthase.
Enzymes: thymidylate synthase (EC 2.1.1.45).
PDB reference: Trichinella spiralis thymidylate synthase-
dUMP complex, 4G9U; Caenorhabditis elegans thymi-
dylate synthase-dUMP complex, 4IRR.
*Corresponding author: Wojciech Rode, Nencki Institute of
Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street,
02-093 Warszawa, Poland, Fax: + 48-22-822-5342,
E-mail: rode@nencki.gov.pl
Anna Dowiercia ł , Piotr Wilk, TomaszFr ą czyk, Adam Jarmu ł a,
Magdalena D ą browska and Joanna Cie ś la: Nencki Institute of
Experimental Biology, Polish Academy of Sciences, Warszawa,
Poland
Wojciech Rypniewski and Katarzyna Banaszak: Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Pozna ń , Poland
Introduction
Thymidylate synthase (TS; EC 2.1.1.45) is an impor-
tant target in chemotherapy, catalyzing the conversion
of deoxyuridine monophosphate (dUMP) and N 5,10 -
methylenetetrahydrofolate (mTHF) to deoxythymidine
monophosphate (dTMP) and dihydrofolate (DHF) [ 1 ].
Enzyme levels in two species of nematodes, parasitic
Trichinella spiralis (causing a serious disease, trichinel-
losis) and free-living Caenorhabditis elegans (considered
a model for parasitic nematodes), have been shown to
remain high throughout the life cycle of each parasite, and
the latter concerned also developmentally arrested, non-
growing larvae, T. spiralis muscle, and C. elegans dauer
larvae [ 2 4 ]. Thus, the enzyme might constitute a poten-
tial target for nematode-selective chemotherapy.
As TS protein, and particularly its active center,
belongs to the most conservative, inhibitors designed
as substrate/cofactor analogs are beyond hope as can-
didates for species-selective inhibitors of the pathogen
versus the mammalian enzyme. 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-con-
servative protein fragments differing between enzymes
from both groups [ 5 ]. To make such an approach pos-
sible, 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 com-
parison with the human enzyme has been so far impos-
sible, as an analogous structure of the non-mutant
human TS-dUMP complex is not available in the Protein
Data Bank. However, in view of high similarity (94.9%)
between mouse and human TS protein sequences, with
88.8% of these sequences being identical, indicated
by the sequence alignment with FASTA [ 7 ], the conclu-
sions derived upon comparison with the mouse enzyme
should hold true for the human enzyme. Further com-
parisons with ternary human and mouse complexes are
planned but these are beyond the scope of the present
paper.
88Dowiercia ł etal.: Crystal structures of nematode thymidylate synthase
Materials and methods
Crystallization and data collection
Each T. spiralis and C. elegans TS recombinant protein, overexpressed
and puri ed as previously described [ 3 , 8 ], was dialyzed against 5mM
Tris HCl bu er, pH 7.5, containing 5mM DTT and then concentrated
using an Amicon Centricon centrifugal  lter. Crystals were grown by
the vapor di usion method in hanging drops at room temperature
( T.spiralis TS) or at 4 ° C ( C. elegans TS). With the T. spiralis enzyme,
3.5 μ L of the protein 20 mg/mL solution, containing 10mM dUMP,
and 2 μ L of the well solution were mixed and allowed to equilibrate
with 0.5mL of the well solution, containing 0.1M Tris HCl, pH 7.9;
0.08M MgCl
2 and 18.5% (w/v) PEG 4000. With the C. elegans enzyme,
2.5 μ L of the protein 23 mg/mL solution, containing 9.5mM dUMP,
and 2.5 μ L of the well solution were mixed and allowed to equilibrate
with 0.5mL of the well solution, containing 0.1M Bis Tris pH 7.2, 0.2M
sodium acetate, 15% PEG 3350.
X-Ray di raction data were collected from a single  ash- frozen
crystal at the BESSY Synchrotron using an X-ray wavelength of
0.918 Å .
Data processing: structure determination
and refinement
Data were processed with the use of DENZO and SCALEPACK [ 9 ]. The
structure was determined by molecular replacement carried out with
Phaser from the CCP4 package [ 10 ], using the mouse TS ternary com-
plex with N 4 -hydoxy-dCMP and DHF as the search model. The cor-
rectness of the structure was evaluated using Sfcheck and Procheck
from the CCP4 suite. Some X-ray data and model re nement param-
eters are presented in Table 1 .
Table 1 Data collection and refinement statistics for structures of Trichinella spiralis, Caenorhabditis elegans , and mouse TS-dUMP binary
complexes.
Crystal and refinement parameters
T. spiralis
TS-dUMP
C. elegans
TS-dUMP
Mouse
TS-dUMP [ 6 ]
Lattice type Triclinic Monoclinic Monoclinic
Space group P P    C   
Unit cell parameters A = . Å a = . Å a = . Å
b = . Å b = . Å b = . Å
c = . Å c = . Å c = . Å
α = . ° α = . ° α = . °
β = . ° β = . ° β = . °
γ = . ° γ = . ° γ = . °
Resolution range, Å . – . . − . . – .
Number of unique reflections , , ,
Redundancy . .
< I/ σ ( Ι ) > . . .
Number of reflections used in refinement , , ,
R factor, % . . .
R free factor, % . . .
RMS bond, Å . . .
RMS angle, ° . . .
Figure 1  Dimer AB of Trichinella spiralis TS-dUMP structure model.
Coloring is according to B-factor values. Both active centers contain
the substrate molecule.
Results and discussion
The structure model of the parasitic nematode T. spiralis
TS complexed with dUMP consists of two dimers (dimer
AB shown in Figure 1 ). It comprises the following amino
acid residues: 17 300/chain A, 17 304/chain B, 18 303/
chain C, and 18 299/chain D. The N- and C-termini, being
not ordered, are not visible in the electron density map.
In each of the four active centers a clear electron density
corresponding to the dUMP molecule is present. The
Dowiercia ł etal.: Crystal structures of nematode thymidylate synthase89
distance between dUMP C6 and catalytic Cys S atoms is
in each subunit longer than 3 Å , pointing to the lack of
covalent bond. The model reveals a high degree of similar-
ity to the mouse structure (model of mTS-dUMP complex;
PDB ID: 4E5O). The C α root mean square deviation
(RMSD) for T. spiralis TS-dUMP/chain A
(22Glu-299Pro) versus
Figure 2  Superimposition of monomers A of Trichinella spiralis
(yellow), Caenorhabditis elegans (green), mouse (pink), and Escher-
ichia coli (cyan) TSs, depicting the substrate molecule, and catalytic
Cys and His ( T. spiralis /mouse TS His190) residues. For each subunit
of the parasitic nematode, TS model His190 can adopt two alterna-
tive conformations; whereas the dominant conformation is similar
to that of the corresponding C. elegans TS and E. coli TS His residue,
the minor one resembles His residue in mouse TS. The image also
shows two conformations of the T. spiralis TS catalytic Cys.
His 190
dominant
Minor
Cys 189
Met 200
Phe 192
Leu 192
Figure 3 Substrate molecule, catalytic Cys189, and the next frag-
ment of three residues of Trichinella spiralis TS (chain A/yellow),
compared with Caenorhabditis elegans TS (green) and mouse TS
(pink). The mouse TS Leu192 corresponds to Phe192 residue in T.
spiralis TS and Met200 residue in C. elegans TS.
mouse TS-dUMP/chain A
(23Gly-299Pro) amounts to 0.798 Å (the
sequence identity for this range being 67.3%). Of inter-
est is that T. spiralis catalytic Cys189 appears capable of
adopting two alternative conformations ( Figures 2 and3).
Also the next residue, His190, appears to acquire two
conformations: the minor one resembles the His confor-
mation in all determined models of mouse enzyme (and
also in human TS model of the complex with dUMP and
Tomudex; PDB ID: 1I00) and the dominant one leans
towards the Tyr224 OH group. The primary His190 confor-
mation in T. spiralis TS is similar to that of the correspond-
ing His conformation in each C. elegans and Escherichia
coli TS (cf. PDB ID: 1BID). Instead of mouse TS Leu192 and
Arg 115
A
B
Val 184
Leu 115
Leu 123
Figure 4 Trichinella spiralis TS (yellow) Arg115, equivalent of
mouse TS (pink) Leu115 and Caenorhabditis elegans (green) Leu123,
hydrogen bonded to Val184. Panel (B) shows the circled fragment of
(A) in magnification.
90Dowiercia ł etal.: Crystal structures of nematode thymidylate synthase
human TS Leu198, the enzyme molecules of T. spiralis and
E. coli contain Phe residue (e.g., T. spiralis Phe192), and
C. elegans TS contains Met (Met200) residue ( Figure 3 ).
Comparing TS active sites, T. spiralis Met206 is replaced
by leucine residue in both C. elegans and mouse TS (cf.
PDB 1BID and 1I00).
Interestingly, the overall similarity between the
C. elegans and mouse enzyme structures appears higher
than that between T. spiralis and mouse TSs. The C α RMSD
for C. elegans TS-dUMP/chain A
(30Asp-307Pro) versus mouseTS-
dUMP/chain A
(23Gly-299Pro) amounts to 0.582 Å . In spite of a
high degree of similarity, superimposing the mouse over
T. spiralis enzyme structures shows approximately 40 sig-
nificant differences with regard to the physicochemical
character of amino acid residues or the distance between
them. In most cases, amino acid substitution results in
rupture of the hydrogen bonding network and is compen-
sated by water molecules. Outside the active site, a sub-
stitution of T. spiralis Arg115 to leucine residue in mouse
(Leu115) and C. elegans (Leu123) TS leads to the loss of two
structurally essential hydrogen bonds, connecting with a
residue (e.g., T. spiralis Val184) from a different part of the
amino acid chain ( Figure 4 ). Another interesting structural
distinction concerns the presence of Cys59 and Phe241 in
the parasitic nematode TS instead of Ser60 and Asp241 in
mouse TS ( Figure 5 ). Together with altered conformation of
the adjacent arginine residue ( T. spiralis Arg61), the two sets
of residues form distinctly different local protein surfaces.
Acknowledgments: This work is supported by the National
Science Centre (grant no. 2011/01/B/NZ6/01781) and the
Ministry of Science and Higher Education (grant no. N301
3948 33).
Received February 28, 2013; accepted March 29, 2013; previously
published online May 7, 2013
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Cie ś la J, etal. Crystal structures of substrate- and sulfate-bound
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dUMP
A
B
Arg 61
Asp 241
Phe 241
Figure 5 Trichinella spiralis TS (yellow) Cys59 and Phe241 are in the
case of mouse and human (structure 1I00) replaced by Ser and Asp
residues, correspondingly. This change involves adjacent arginine
residue conformation modification. Panel (B) shows the circled frag-
ment of (A) in magnification.
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... Each purified T. spiralis and C. elegans TS recombinant protein was dialyzed against 5 mM Tris HCl buffer, pH 7.5, containing 5 mM DTT, and concentrated using an Amicon Centricon centrifugal filter (cf. [67]). Crystals were grown by the vapor diffusion method in hanging drops at room temperature (TsTS) or at 4 • C (CeTS). ...
... As a consequence of Tyr260 and His258 involvement in hydrogen bonding of the 3 -OH group of the deoxyribose moiety of dUMP, this segment progressively shifts toward the active site. The effect is initiated upon binding of dUMP in the binary complex [67] and reinforced upon binding of Raltitrexed in the ternary complex. Finally, a strictly specific effect of the binding of Raltitrexed is apparent for the AA6 helix-loop segment Asp116-Gly131, localized on the opposite flank of the entrance to the active site pocket (in relation to the loop Arg48-Gly55) and shifted toward the active site in the CeTS-dUMP-Raltitrexed complex, but not upon the binding of dUMP in the CeTS-dUMP complex, where this segment moves very slightly and nearly retains its position from the ligand-free structure. ...
... The distances between C6 of dUMP and S of Cys197 rule out the possibility of a covalent linkage between the substrate and enzyme in both the CeTS-dUMP-Raltitrexed (3.5 Å on average) and the CeTS-dUMP (3.3 Å) [67] structures. In accord, the dUMP molecule occupies the same position in both ligand-bound structures. ...
... 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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We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
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Localization of thymidylate synthase protein in Trichinella spiralis and Caenorhabditis elegans development was followed with the use of confocal microscopy, revealing similar expression patterns in both nematode species. In T. spiralis premature muscle larvae and C. elegans dauer, L3 and L4 larvae, thymidylate synthase was detected in the nerve ring and gonad primordia, as well as T. spiralis stichosome and C. elegans pharyngeal glandular cells. In developmentally arrested T. spiralis muscle larvae, the enzyme was found localized to the gonad primordia and stichosome. High enzyme level was also observed in the embryos developing in uteri of T. spiralis female adult and C. elegans hermaphrodite forms. In the case of T. spiralis adult forms, thymidylate synthase was detected in stichosome, along esophagus wall, as well as in egg and sperm cells. While the enzyme protein present in the embryos remains in accord with its known association with proliferating systems, thymidylate synthase presence in the nerve ring, and reproductive and secretory (T. spiralis stichosomal and C. elegans pharyngeal glandular cells) systems, points to a state of cell cycle-arrest, also known to preserve the enzyme protein.
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Thymidylate synthase is a target enzyme in anticancer, antiviral, antifungal and antiprotozoan chemotherapy. With two dUMP analogues, 5-fluoro-dUMP (FdUMP) and 5-(trifluoromethyl)-dUMP (CF3dUMP), strong thymidylate synthase inhibitors and active forms of drugs, the inhibition mechanism is based on the reaction mechanism. Recent comparative studies of new dUMP analogues, containing more than one substituent in the pyrimidine ring, showed that substitution of the pyrimidine ring C(4) = O group in FdUMP by either C(4) = N-OH group (in N4-hydroxy-FdCMP) or C(4) = S group (in 4-thio-FdUMP) preserves high inhibitory potency of the drug but may alter its specificity for thymidylate synthases from various sources, which differ in sensitivity to slow-binding inhibition by FdUMP. Informations suggesting mechanisms responsible for the foregoing have been reviewed, including results of molecular modeling studies suggesting interaction of the pyrimidine C(4) = O group, or its modification, with the N5,10-methylene.(ABSTRACT TRUNCATED)