THE PLASMODIUM GLUTATHIONE S-TRANSFERASE

Bioinformatics Characterization and Classification into the Sigma Class

Emilee E. Colón-Lorenzo, Adelfa E. Serrano

Department of Microbiology and Medical Zoology, University of Puerto Rico School of Medicine, San Juan, Puerto Rico

Hugh B. Nicholas Jr, Troy Wymore, Alexander J. Ropelewski

Pittsburgh Supercomputing Center, Pittsburgh, PA, U.S.A.

Ricardo González-Méndez

Department of Radiological Sciences, University of Puerto Rico School of Medicine, San Juan, Puerto Rico

Keywords: Malaria, Plasmodium, Glutathione S-transferase, Bioinformatics analysis, Structural alignment, GST

classification, Structural modeling.

Abstract: Malaria is a global health problem caused by Plasmodium parasites. Glutathione S-transferase (GST) is

involved in the conjugation of glutathione to drugs and toxic compounds. It is postulated that GST plays an

important role in the development of drug resistance. The three-dimensional (3D) structure of Plasmodium

falciparum GST (PfGST) has been solved and previous work indicates that the PfGST cannot be assigned to

any of the known GST classes. We performed sequence analyses, structural modeling and alignment of

GSTs from Plasmodium to known structures of the GST from other organisms to classify PfGST into a GST

family. Sequence alignments using ClustalW, motif analysis using MEME, and phylogenetic analysis using

MEGA4, of Plasmodium GSTs and 38 other GST sequences were done. The alignments and motifs show a

close relationship to the alpha and sigma class of GSTs. The phylogenetic analysis places the Plasmodium

GSTs in the sigma class. A comparison of PfGST with known structures of GSTs reveals high structural

similarity to the sigma class GST, in particular within the H-site and C-terminus of the protein. These

findings allow PfGST to be classified into the sigma class GSTs. These data may open new avenues for the

development of novel antimalarials.

1 INTRODUCTION

Malaria is one of the most devastating diseases in

the world caused by parasites of the genus

Plasmodium. It is estimated that more than 350

million cases of malaria infections and, over one

million deaths occur annually (WHO, 2005). The

inappropriate and indiscriminate use of drugs has led

to the development of drug resistance in parasites

(Russell, 2004; Whitty et al., 2002; Wongsrichanalai

et al., 2002). Efficient and cost effective alternatives

to presently used drugs are not yet available. A

comprehensive understanding of parasite

development and drug resistance will enable the

development of more effective drug therapies to

combat this disease.

Glutathione S-transferases (GSTs) are a family

of detoxification enzymes found in most organisms

that conjugate reduced glutathione (GSH) with toxic

electrophilic organic compounds and drugs. GSTs

have been subdivided into different classes based on

their primary structure, immunological properties

and substrate specificities (Winayanuwattikun and

Ketterman, 2005). The GST classes are widespread

and are present in a variety of organisms. These

classes include the following: alpha, sigma, mu, pi,

theta, zeta and omega classes (Torres-Rivera and

Landa, 2008). Additionally, there are organism-

specific classes which include several GSTs which

are found only in certain kingdoms or phyla:

lambda, phi, and tau in plants; delta, epsilon in

173

E. Colón-Lorenzo E., E. Serrano A., B. Nicholas Jr H., Wymore T., J. Ropelewski A. and González-Méndez R. (2010).

THE PLASMODIUM GLUTATHIONE S-TRANSFERASE - Bioinformatics Characterization and Classiﬁcation into the Sigma Class.

In Proceedings of the First International Conference on Bioinformatics, pages 173-180

DOI: 10.5220/0002747401730180

 SciTePress

insects; and beta in prokaryotes. Furthermore, the

kappa class is a unique mitochondrial GST.

Altered GSH mediated detoxification is one of

the proposed underlying mechanisms for the

development of malaria drug resistance. A marked

increased in both GSH levels and GSH-related

enzymatic activity was reported in P. berghei and P.

falciparum lines resistant to chloroquine (Dubois et

al., 1995; Meierjohann et al., 2002; Srivastava et al.,

1999b). Recent results showed that the glutathione

biosynthesis is essential for the parasite’s life cycle

and mosquito transmission (Vega-Rodríguez et al.,

2009). These are some of the reasons why GST is

considered a very promising drug target for the

design of antimalarial drugs.

The genomes of P. falciparum (Pf), P. vivax

(Pv), P. knowlesi (Pk) and P. y. yoelii (Py) have

been sequenced (Carlton et al., 2008) and revealed

that the parasite harbors only one GST. In addition,

the three-dimensional (3D) structure of PfGST has

been solved (Burmeister et al., 2003; Fritz-Wolf et

al., 2003; Perbandt et al., 2004). Detailed structural

comparison to representative structures of the alpha,

mu, and pi classes indicate that the PfGST cannot be

assigned to any of the above GST classes (Fritz-

Wolf et al., 2003; Deponte and Becker, 2005). It was

found that PfGST adopts the canonical GST fold and

is enzymatically active as a homodimer. All the

known structures of GSTs reveal a similar overall

fold: a homodimer where each monomer contains an

N-terminal α/β-domain with βαβαββα topology and

a C-terminal α-helical domain. The active site of

GST is located between the two domains. The active

site possesses two binding sites: the G-site, which

binds reduced GSH, and the H-site, which can

accommodate a variety of substrates. In PfGST, the

N-terminal domain possesses the G-site similar to

that of the alpha, mu, and pi classes studied. The C-

terminal domain shields the H-site. Also, the H-site

in PfGST differed from the other GSTs studied. But

more importantly, PfGST only has 5 amino acid

residues after alpha helix 8 and cannot form the

required structural elements of the alpha, mu, and pi

classes (Fritz-Wolf

et al., 2003).

Here we report bioinformatics sequence analyses

and structural modeling of GSTs from Plasmodium.

We performed a phylogenetic analysis of

Plasmodium spp. sequences and 38 other GST

sequences. We have also analyzed the 3D structure

of PfGST to classify the protein into a GST family.

Our results using PfGST suggest that the

Plasmodium GSTs are a unique family related to the

sigma class. This work is significant for the future

design of specific inhibitors for Plasmodium GSTs

which may lead to the development of novel

antimalarials.

2 MATERIALS AND METHODS

2.1 Alignment of the Plasmodium GSTs

The GST sequences recovered from Plasmodium

spp. and used for subsequent sequence analysis are

listed in Table 1 in the Appendix. The amino acid

sequences were aligned using ClustalW program

(Higgins et al., 1994) with the default parameters.

The alignment was visualized using GeneDoc

program (Nicholas et al., 1997) and some manual

editing was made to produce the final alignment.

2.2 Classification of P. falciparum GST

by Alignment and Phylogenetic

Analysis

We performed a BLAST search with PfGST

(Q8MU52) as the query sequence, using the

iProClass database (Wu et al., 2003). The BLAST

search was carried out using the default parameters.

We selected five members of each of the GST

classes (alpha, sigma, pi, mu, delta, tau and theta)

except for the zeta class where three sequences were

chosen. Since Plasmodium is an eukaryotic

organism, we focused on sequences from mammals,

plants and insects, excluding prokaryotic GSTs. The

GST sequences used are listed in Table 1 in the

Appendix. The multiple sequence alignment was

performed using ClustalW (Higgins et al., 1994).

The Multiple Entropy for Motif Elicitation (MEME)

program was used to find 20 conserved motifs using

‘zero or more occurrences per sequence’ pattern for

selection of motifs (Bailey et al., 1994). The

alignment and motifs were visualized with GeneDoc

(Nicholas et al., 1997). The multiple sequence

alignment was trimmed manually and then used to

perform the phylogenetic analysis. The phylogenetic

analysis was done using the MEGA4 program

(Tamura et al., 2007) using the neighbor-joining

algorithm and a bootstrapped data set of 100

replicates. The program FigTree

(http://tree.bio.ed.ac.uk/software/figtree) was used to

visualize the consensus tree from the bootstrap

analysis.

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2.3 Classification of P. falciparum GST

by Structural Alignment

Structural alignment of P. falciparum GST (1Q4J)

with a representative member of each of the GST

classes - alpha, sigma, pi and mu - was performed

using the MultiSeq feature in VMD (Roberts et al.,

2006, Humphrey et al., 1996). The 3D structures

were obtained from the Protein Data Bank (PDB)

(Berman et al., 2000) and are listed in Table 2 in the

Appendix. The sigma GST from Onchocerca

volvulus (2HNL) and the human alpha GST (1PKZ)

were used to perform the structural alignments with

PfGST (shown in Figure 4). The detailed structural

superpositions were carried out with VMD,

specifically in the N-terminal domain (G-site), C-

terminal domain (H-site), and α-helix at the C-

terminus of the proteins. Close-up views of the H-

site, G-site and α-helix in the C-terminus were done

to facilitate the analysis of the 3D structure of the

proteins.

3 RESULTS AND DISCUSSION

3.1 Alignment of the Plasmodium GSTs

The multiple sequence alignment of sequences from

Plasmodium spp. GST is shown in Figure 1. The

sequences used in the alignment are listed in Table 1

in the Appendix. The alignment of Plasmodium spp.

GSTs revealed a significant degree of sequence

identity ranging from 80 to 87%.

Figure 1: Multiple sequence alignment of the Plasmodium

GSTs. Amino acids with a 100% identity are shaded in

red, 99-80% identity are in blue and 79-60% identity in

green.

The GST sequences are highly conserved in all four

species of Plasmodium.

3.2 Classification of P. falciparum GST

by Alignment and Phylogenetic

Analysis

A BLAST search in iProClass was performed using

GST from P. falciparum (Q8MU52). We selected

five sequences of each GST classes except for zeta

class where we selected only three sequences. A

total of 38 GST sequences were obtained and are

listed in Table 1 in the Appendix. The multiple

sequence alignment was done using the program

ClustalW and the MEME program was used to find

20 conserved motifs (Figure 2). The multiple

sequence alignment and motif analyses show that the

Plasmodium GSTs appear to be highly related to the

alpha and sigma families of GST. The alignment

was trimmed manually and was used to perform the

phylogenetic analysis (Figure 3). The phylogenetic

tree indicates that Plasmodium spp. GSTs analyzed

are members of the sigma class of GSTs.

3.3 Classification of P. falciparum GST

by Structural Alignment

The three-dimensional structure superimposition

makes possible the classification of P. falciparum

glutathione S-transferase in a specific GST class.

The three-dimensional structures of GST enzymes

from various classes (alpha, sigma, mu and pi) were

compared to the PfGST 3D structure (1Q4J) by

structural alignment using the MultiSeq feature in

the VMD program. The 3D structures used in the

structural alignments are listed in Table 2 in the

Appendix. Structural alignments of PfGST 3D

structure (1Q4J) with alpha and sigma 3D structures

are presented in Figure 4.

Figure 4A shows the structural alignment of PfGST

(1Q4J) with sigma GST from Oncocherca volvulus

(2HNL). A close-up view showing the α-helix of the

C-terminus is represented in Figure 4B. Figure 4C

shows the structural alignment of PfGST (1Q4J)

with the alpha GST from human (1PKZ). A close-up

view showing the C-terminus is represented in

Figure 4D. Analysis of the 3D structural alignment

of PfGST with the sigma class GST structure shows

high structural similarity in the C-terminus (Figure

4B). The 3D structural alignment of PfGST and the

alpha GST demonstrates a good alignment, but the

C-terminus shows low similarity (Figure 4C). In

addition, we can see that PfGST does not have the

extended helix in the C-terminus that is distinctive

of the alpha class of GST (Figure 4D). Structural

alignments of PfGST 3D structure (1Q4J) with mu

and pi 3D structures were performed showing a low

THE PLASMODIUM GLUTATHIONE S-TRANSFERASE - Bioinformatics Characterization and Classification into the

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Figure 2: Schematic sequence alignment of four Plasmodium GSTs and 38 GST sequences from eight known GST classes.

Double lines represent where amino acids are present and single lines represent gaps in the alignment. MEME motifs are

identified by different colors and adjusting edges of MEME motifs was the major alignment adjustment. Plasmodium

sequences are colored in orange, alpha sequences in cyan, sigma sequences in purple, pi sequences in red, mu sequences in

green, delta sequences in blue, tau sequences in magenta, zeta sequences in olive green and theta sequences in yellow on the

left side of the figure.

Figure 3: Phylogenetic tree for GST sequences with the fraction of the bootstrap. Plasmodium (orange), sigma (purple),

alpha (cyan), pi (red), mu (green), delta (blue), tau (magenta), zeta (olive green) and theta (yellow).

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Figure 4: Structural alignment of P. falciparum GST with

sigma and alpha class GST structure. (A) Structural

alignment of PfGST (cyan) and a sigma class GST (blue);

(B) Close-up view showing the C-terminus of PfGST

(yellow) and sigma GST (red); (C) Structural alignment of

PfGST (cyan) and an alpha class GST (blue); (D) Close-

up view showing the C-terminus of PfGST (yellow) and

alpha GST (red).

structural similarity (data not shown). An important

feature of the C-terminus of alpha class GST is an

alpha helix close to the active site. This helix is an

essential element of the GST alpha class (Nilsson et

al., 2002). Based on this, we performed an analysis

of this area of the protein using structural

alignments. Fritz-Wolf et al. (2003) described the

PfGST having a different length C-terminus - only

has 5 amino acid residues after alpha helix 8 - and

therefore could not form the required structural

elements from mammalian GSTs as part of what

makes them not classifiable. Our observation is that

PfGST aligned well with the sigma class GST from

O. volvulus. We find that using common structural

features and MEME patterns GSTs have a very

consistent C-terminus length within each class and

that is one of the features that helped clarify the

classification of the Plasmodium sequences as a

sigma class in both the structural alignment (Figure

4) and the sequence alignment (Figure 2).

Furthermore, analysis of the 3D structure of

PfGST in the N-terminal domain (G-site) and C-

terminal domain (H-site) was performed. Structural

alignment of the N-terminal domain, that contains

the G-site, reveals that PfGST shares a common

backbone fold with the sigma (2HNL) GST from O.

volvulus and alpha (1PKZ) GST from human. Both

structure alignments show a similar binding mode

for S-hexylglutathione (GSH derivative). Close

views of the N-terminal domain are shown (Figures

5A and 5C, respectively). Structural alignment of the

C-terminal domain, that contains the H-site, shows

that PfGST shares a common backbone with sigma

(2HNL) GST, while alpha (1PKZ) GST is different,

specifically in the C-terminus (Figures 5B and 5D,

respectively). Generally, the H-site of GST enzymes

is more variable than the G-site due to the great

number of secondary structures (Fritz-Wolf et al.,

2003). These findings are in agreement with Fritz-

Wolf et al. (2003).

Figure 5: Structural comparison of GST structures from P.

falciparum GST with a sigma GST from O. volvulus and

an alpha GST from human. (A) Structural alignment of N-

terminal (G-site) of PfGST (cyan) and a sigma class GST

(blue); (B) Structural alignment of C-terminal (H-site) of

PfGST (cyan) and a sigma class GST (blue); (C)

Structural alignment of N-terminal (G-site) of PfGST

(cyan) and a alpha class GST (blue); (D) Structural

alignment of C-terminal (H-site) of PfGST (cyan) and a

alpha class GST (blue). The S-hexylglutathione ligand is

shown as a ball-and-stick model.

A comprehensive analysis of the 3D structure of

PfGST in the H-site, G-site and C-terminus of the

proteins was done and established that these regions

have high similarity with the sigma class GST from

O. volvulus (2HNL). The structural alignments

support the results obtained with the phylogenetic

tree. These results allow us to classify the

Plasmodium GSTs as members of the sigma class of

GSTs.

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4 CONCLUSIONS

The GST sequences of four Plasmodium species

were compared using multiple sequence alignments

and found to be highly conserved. When aligned

with members of each of the GST classes used in

this study our results show that the Plasmodium

GSTs appear to be highly related to the alpha and

sigma families of GSTs. Using phylogenetic analysis

we found that Plasmodium GST are members of the

sigma class. The 3D structure of PfGST was

compared, using structural alignments, with

members of several GST classes. We determined

that PfGST has a high degree of similarity with the

sigma class in the H-site, G-site and C-terminus of

the protein. Structural analysis and phylogenetic

analysis of PfGST revealed that this enzyme

represents a unique clade within the sigma class of

GSTs.

This work contributes to a better understanding

of the structure and classification of Plasmodium

GSTs. These results have the potential to enhance

our knowledge of the relevance of GST to

Plasmodium drug resistance. It has been recently

demonstrated that 7-nitro-2,1,3-benzoxadiazole

derivatives are a new class of suicide inhibitors of

GST that accumulate in tumor cells and evade the

extrusion mechanisms mediated by the multidrug

resistance associated protein pumps (MRP) (Ricci et

al., 2005). Studies in human cancer cells in-vitro

showed that the GST inhibitor 6-(7-nitro-2,1,3-

benzoxadiazol-4-ylthio)hexanol (NBDHEX) has

high antiproliferative activity and helped overcome

MRP mediated drug resistance (Federici et el., 2009;

Filomeni et al., 2008). In-silico studies will be

undertaken to evaluate NBDHEX’s potential as an

antimalarial drug. These studies could support the

use of this GST inhibitor in Plasmodium.

We expect to use the results presented here for

further studies using molecular biology and genetics

approaches in order to study the involvement of

GST in the Plasmodium life cycle and mosquito

transmission. These studies should be useful to find

alternative strategies for malaria control.

ACKNOWLEDGEMENTS

RGM, EECL, HBN, TW, AJP and this project were

partially supported by NIGMS grant T36-

GM008789 and acknowledge use of the Pittsburgh

Supercomputing Center’s National Resource for

Biomedical Supercomputing resources funded

through NIH NCRR grant P41-RR06009. AES and

EECL were partially supported by NIGMS/MBRS

award GM08224 and from the RCMI award

G12RR03051 of the NCRR/NIH.

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APPENDIX

Table 1: Glutathione S-transferase sequences used in the alignments.

Protein name Organism Family class Accession number Protein length (aa)

PfGST Plasmodium falciparum sigma Q8MU52 211

PvGST Plasmodium vivax sigma Q0ZS46 205

PkGST Plasmodium knowlesi sigma B3LAI5 205

PyGST Plasmodium yoelii sigma Q7REH6 209

GSTA1_HUMAN Homo sapiens alpha P08263 222

GSTA1_RAT Rattus norvegicus alpha P00502 222

GSTA1_MOUSE Mus musculus alpha P13745 223

GSTA1_PIG Sus scrofa alpha P51781 222

GSTA1_CAVPO Cavia porcellus alpha P81706 218

GST1_ONCVO Oncocherca volvulus sigma P46434 235

GST_MUSDO Musca domestica sigma P46437 241

GST_OMMSL Ommastrephes sloanei sigma P46088 203

GST4_CAEEL Caenorhabditis elegans sigma Q21355 207

GST3_CAEEL Caenorhabditis elegans sigma O16116 207

GSTP1_HUMAN Homo sapiens pi P09211 210

GSTP1_MOUSE Mus musculus pi P19157 210

GSTP1_RAT Rattus norvegicus pi P04906 210

GSTP1_PIG Sus scrofa pi P80031 207

GSTP1_BOVIN Bos taurus pi P28801 210

GSTM1_HUMAN Homo sapiens mu P09488 218

GSTM1_RAT Rattus norvegicus mu P04905 218

GST26_FASHE Fasciola hepatica mu P30112 218

GSTM1_MOUSE Mus musculus mu P10649 218

GSTMU_RABIT Oryctolagus cuniculus mu P46409 218

GST1D_ANOGA Anopheles gambiae delta Q93113 209

GSTT2_MUSDO Musca domestica delta P46431 210

GSTT3_MUSDO Musca domestica delta P46432 210

GSTT5_DROME Drosophila melanogaster delta Q9VG95 216

GSTT4_DROME Drosophila melanogaster delta Q9VG96 215

O24595_MAIZE Zea mays tau O24595 224

O81602_MESCR Mesembryanthemum crystallinum tau O81602 224

Q9ZRW8_ARATH Arabidopsis thaliana tau Q9ZRW8 219

Q43678_9FABA Vigna radiate tau Q43678 230

O49821_CARPA Carica papaya tau O49821 218

GSTZ-WHEAT Triticum aestivum zeta O04437 213

GSTZ1_DIACA Dianthus caryophyllus zeta P28342 221

GSTZ_EUPES Euphorbia esula zeta P57108 225

GSTT1_MOUSE Mus musculus theta Q64471 240

GSTT2_MOUSE Mus musculus theta Q61133 244

GSTT1_RAT Rattus norvegicus theta Q01579 240

GSTT1_CHICK Gallus gallus theta P20135 261

GSTT2_HUMAN Homo sapiens theta P30712 244

Table 2: Glutathione S-transferase sequences used for the structural alignments.

Protein name Organism Family class PDB code

PfGST Plasmodium falciparum sigma 1Q4J

GST1_ONCVO Oncoherca volvulus sigma 2HNL

DmGST Drosophila melanogaster sigma 1MOU

GSTA1_HUMAN Homo sapiens alpha 1PKZ

GSTM1_HUMAN Homo sapiens mu 1GTU

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