Native and Inhibited Structure of a Mu class-related Glutathione S-transferase from Plasmodium falciparum

Abstract

The parasite Plasmodium falciparum causes malaria tropica, the most prevailing parasitic disease worldwide, with 300 -500 million infections and 1.5-2.7 million deaths/year. The emergence of strains resistant to drugs used for prophylaxis and treatment and no vaccine available makes the structural analysis of potential drug targets essential. For that reason, we analyzed the three-dimensional structure of the glutathione S-transferase from P. falciparum (Pf-GST1) in the apoform and in complex with its inhibitor S-hexyl-glutathione. The structures have been analyzed to 2.6 and 2.2 Å, respectively. Pf-GST1 shares several structural features with the Mu-type GSTs and is therefore closely related to this class, even though alignments with its members display low sequence identities in the range of 20 -33%. Upon S-hexyl-glutathione binding, the overall structure and the glutathione-binding site (G-site) remain almost unchanged with the exception of the flexible C terminus. The detailed comparison of the parasitic enzyme with the human host Mu-class enzyme reveals that, although the overall structure is homologue, the shape of the hydrophobic binding pocket (H-site) differs substantially. In the human enzyme, it is shielded from one side by the large Mu-loop, whereas in Pf-GST1 the Mu-loop is truncated and the space to recognize and bind voluminous substrates is extended. This structural feature can be exploited to support the design of specific and parasite-selective inhibitors.Sophisticated defense strategies have evolved in organisms that enable them to deal with a broad range of foreign and endogenously derived toxic compounds. These structurally diverse usually highly non-polar molecules are by definition not required for normal metabolic functions, and enzyme systems that catalyze the rapid conversion of these compounds have been developed. However, instead of converting these compounds to useful metabolites as described for a vast array of highly specific enzymes among a large number of bacteria, higher life forms have opted for elimination of toxic compounds rather than their utilization (1). Generally, the pathways of detoxification can be divided into three phases. Phase I reactions consist of the so-called "functionalization" reactions, where a reactive group is demasked or introduced into the molecule. These reactive groups then can be conjugated with natural constituents of the organism (phase II) prior to excretion (phase III) (2).Glutathione S-transferases (GSTs, 1 EC 2.5.1.18) are a family of phase II detoxification enzymes found in most organisms. Eucaryotes usually contain multiple GSTs with different catalytic activities to accommodate a wide range of functions within the cells. Cytosolic GSTs of mammals are divided into seven classes named Alpha, Mu, Pi, Theta, Zeta, Sigma, and Omega on the basis of different amino sequences and substrate specificities. Additionally, the Kappa GST is a soluble mitochondrial enzyme. Studies of GSTs from non-mammalian species have revealed the existence of several ne...

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113–119

“…The basic procedure utilized the purified expression vector pJC20 containing the complete cDNA of the PfGST as an insert (18) and two synthetic oligonucleotide primers designed to introduce the proper mutations. In mutant A, the flexibility of the loop region was lowered by the substitution of Thr-113 and Asn-119 by two prolines.…”

“…Thus, in these mutants the inactivation process becomes at least 100 times slower than in the native enzyme. Recent studies based on gel filtration experiments have demonstrated that the inactivation caused by the absence of GSH is coupled to a tetramerization process (16,18). This Tetramerization and Cooperativity in P. falciparum GST AUGUST 14, 2009 • VOLUME 284 • NUMBER 33…”

“…The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13,18), provides insights into this effect. From a structural point of view, the most intriguing differences of PfGST when compared with other GSTs are a more solvent-exposed H-site and an atypic extra loop connecting helix ␣-4 and helix ␣-5 (residues 113-119; see also Fig.…”

“…Actually, in the absence of ligands, two biological dimers form a tetramer, and these homodimers are interlocked with each other by loop 113-119 of one homodimer, which occupies an H-site of the other homodimer (13,18). Upon binding of S-hexylglutathione, loop 113-119 rearranges; residues Asn-114, Leu-115, and Phe-116 form an additional coil in helix ␣-4; and the side chains of Asn-111, Phe-116, and Tyr-211 flip into the H-site of the same dimer (17,18). The changed course of residues 113-119 in the liganded enzyme prevents the interlocking of the dimers.…”

Cooperativity and Pseudo-cooperativity in the Glutathione S-Transferase from Plasmodium falciparum

“…A gene encoding a protein with sequence identity of up to 33% with known GSTs was identified on chromosome 14 of P. falciparum. The three-dimensional structure of PfGST shares several structural features with the Mu class GSTs even though alignments with its members display low sequence identities (20 -33%) (12). Despite this structural similarity with Mu class GSTs, PfGST shows very low or no affinity toward the classical GST substrates.…”