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

Anna Dowierciał; Piotr Wilk; Wojciech Rypniewski; Tomasz Frączyk; Adam Jarmuła; Katarzyna Banaszak; Magdalena Dąbrowska; Joanna Cieśla; Wojciech Rode

doi:10.1515/pterid-2013-0011

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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

Anna Dowierciał , Piotr Wilk , Wojciech Rypniewski , Tomasz Frączyk , Adam Jarmuła , Katarzyna Banaszak , Magdalena Dąbrowska , Joanna Cieśla und Wojciech Rode

Veröffentlicht/Copyright: 7. Mai 2013

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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.

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 thymidylate synthase-dUMP complex, 4IRR

Introduction

Thymidylate synthase (TS; EC 2.1.1.45) is an important 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, trichinellosis) 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 potential 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 candidates 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-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. 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 conclusions derived upon comparison with the mouse enzyme should hold true for the human enzyme. Further comparisons with ternary human and mouse complexes are planned but these are beyond the scope of the present paper.

Materials and methods

Crystallization and data collection

Each T. spiralis and C. elegans TS recombinant protein, overexpressed and purified as previously described [3, 8], was dialyzed against 5 mM Tris HCl buffer, pH 7.5, containing 5 mM DTT and then concentrated using an Amicon Centricon centrifugal filter. Crystals were grown by the vapor diffusion method in hanging drops at room temperature (T. spiralis TS) or at 4°C (C. elegans TS). With the T. spiralis enzyme, 3.5 μL of the protein 20 mg/mL solution, containing 10 mM dUMP, and 2 μL 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 MgCl₂ and 18.5% (w/v) PEG 4000. With the C. elegans enzyme, 2.5 μL of the protein 23 mg/mL solution, containing 9.5 mM dUMP, and 2.5 μL 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 diffraction data were collected from a single flash-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 complex with N⁴-hydoxy-dCMP and DHF as the search model. The correctness of the structure was evaluated using Sfcheck and Procheck from the CCP4 suite. Some X-ray data and model refinement parameters 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	P1	P 32 2 1	C 1 2 1
Unit cell parameters	A=51.695 Å	a=135.09 Å	a=160.35 Å
	b=65.914 Å	b=135.09 Å	b=88.54 Å
	c=96.511 Å	c=155.77 Å	c=136.76 Å
	α=85.305°	α=90.00°	α=90.00°
	β=85.327°	β=90.00°	β=95.99°
	γ=67.117°	γ=120.00°	γ=90.00°
Resolution range, Å	19.96–1.90	29.2−2.48	19.95–1.70
Number of unique reflections	89,350	58,033	207,806
Redundancy	5	6.4	6.4
<I/σ(Ι)>	6.1	8.1	16.9
Number of reflections used in refinement	89,301	55,056	205,349
R factor, %	16.5	22.4	23.6
R_free factor, %	22.0	26.3	29.2
RMS bond, Å	0.022	0.0117	0.022
RMS angle, °	1.834	1.5183	1.956

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 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 similarity to the mouse structure (model of mTS-dUMP complex; PDB ID: 4E5O). The Cα root mean square deviation (RMSD) for T. spiralis TS-dUMP/chain A_{(22Glu-299Pro)} versus mouse TS-dUMP/chain A_{(23Gly-299Pro)} amounts to 0.798 Å (the sequence identity for this range being 67.3%). Of interest 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 conformation 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 conformation in T. spiralis TS is similar to that of the corresponding His conformation in each C. elegans and Escherichia coli TS (cf. PDB ID: 1BID). Instead of mouse TS Leu192 and 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).

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.

Figure 2

Superimposition of monomers A of Trichinella spiralis (yellow), Caenorhabditis elegans (green), mouse (pink), and Escherichia 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 alternative 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 fragment 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.

Interestingly, the overall similarity between the C. elegans and mouse enzyme structures appears higher than that between T. spiralis and mouse TSs. The Cα 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 significant 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 compensated by water molecules. Outside the active site, a substitution 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.

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.

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 fragment of (A) in magnification.

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

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).

References

1. Rode W, Leś A. Molecular mechanism of thymidylate synthase-catalyzed reaction and interaction of the enzyme with 2- and/or 4-substituted analogues of dUMP and 5-fluoro-dUMP. Acta Biochim Pol 1996;43:133–42.10.18388/abp.1996_4524Suche in Google Scholar

2. Dąbrowska M, Jagielska E, Cieśla J, Płucienniczak A, Kwiatkowski J, Wranicz M, et al. Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression. Parasitology 2004;128:209–21.10.1017/S0031182003004426Suche in Google Scholar PubMed

3. Wińska P, Gołos B, Cieśla J, Zieliński Z, Frączyk T, Wałajtys-Rode E, et al. Developmental arrest in C. elegans dauer larvae leaves high expression of enzymes involved in thymidylate biosynthesis, similar to that found in Trichinella muscle larvae. Parasitology 2005;131:247–54.10.1017/S0031182005007274Suche in Google Scholar PubMed

4. Gołos B, Dąbrowska M, Wałajtys-Rode E, Zieliński Z, Wińska P, Cieśla J, et al. Immunofluorescent localization of thymidylate synthase in the development of Trichinella spiralis and Caenorhabditis elegans. Mol Biochem Parasitol 2012; 183:63–9.10.1016/j.molbiopara.2012.02.002Suche in Google Scholar PubMed

5. Shoichet BK, Stroud RM, Santi DV, Kuntz ID, Perry KM. Structure-based discovery of inhibitors of thymidylate synthase. Science 1993;259:1445–50.10.1126/science.8451640Suche in Google Scholar PubMed

6. Dowierciał A, Jarmuła A, Rypniewski W, Sokołowska M, Frączyk T, Cieśla J, et al. Crystal structures of substrate- and sulfate-bound mouse thymidylate synthase. Pteridines 2009;20:163–7.Suche in Google Scholar

7. Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 1988;85:2444–8.10.1073/pnas.85.8.2444Suche in Google Scholar PubMed PubMed Central

8. Frączyk T, Ruman T, Rut D, Dąbrowska-Maś E, Cieśla J, Zieliński Z, et al. Histidine phosphorylation, or tyrosine nitration, affect thymidylate synthase properties. Pteridines 2009; 20:137–42.Suche in Google Scholar

9. Otwinowski Z, Minor W. Processing of X-ray diffraction data collected in oscillation mode. In: Carter CW Jr, Swee RM, editors. Methods in enzymology. Macromolecular crystallography, vol. 276, part A. New York: Academic Press, 1997:307–26.Suche in Google Scholar

10. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Cryst 1994;D50:760–3.10.1107/S0907444994003112Suche in Google Scholar PubMed

Received: 2013-2-28

Accepted: 2013-3-29

Published Online: 2013-05-07

Published in Print: 2013-06-01

This article is distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Artikel in diesem Heft

https://doi.org/10.1515/pterid-2013-0011

Schlagwörter für diesen Artikel

3D structure; nematode; thymidylate synthase; Enzymes: thymidylate synthase (EC 2.1.1.45); PDB reference: Trichinella spiralis thymidylate synthase-dUMP complex, 4G9U; Caenorhabditis elegans thymidylate synthase-dUMP complex, 4IRR

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