DOI 10.1515/pterid-2013-0011Pteridines 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
templatefor 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 220.127.116.11).
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,
Anna Dowiercia ł , Piotr Wilk, TomaszFr 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,
Wojciech Rypniewski and Katarzyna Banaszak: Institute of
Bioorganic Chemistry, Polish Academy of Sciences, Pozna ń , Poland
Thymidylate synthase (TS; EC 18.104.22.168) 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
88Dowiercia ł etal.: 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 5mM
Tris HCl bu er, pH 7.5, containing 5mM 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 10mM dUMP,
and 2 μ L of the well solution were mixed and allowed to equilibrate
with 0.5mL of the well solution, containing 0.1M Tris HCl, pH 7.9;
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 9.5mM dUMP,
and 2.5 μ L of the well solution were mixed and allowed to equilibrate
with 0.5mL of the well solution, containing 0.1M Bis Tris pH 7.2, 0.2M
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
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
Crystal and refinement parameters
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 etal.: Crystal structures of nematode thymidylate synthase89
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
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.
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 and3).
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
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.
90Dowiercia ł etal.: 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-
(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
Received February 28, 2013; accepted March 29, 2013; previously
published online May 7, 2013
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