Glutathione mediated regulation of oligomeric structure and functional activity of Plasmodium falciparum glutathione S-transferase

Abstract

Background: In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one typical glutathione S-transferase. This enzyme, PfGST, cannot be assigned to any of the known GST classes and represents a most interesting target for antimalarial drug development. The PfGST under native conditions forms non-covalently linked higher aggregates with major population (approximately 98%) being tetramer. However, in the presence of 2 mM GSH, a dimer of PfGST is observed. Recently reported study on binding and catalytic properties of PfGST indicated a GSH dependent low-high affinity transition with simultaneous binding of two GSH molecules to PfGST dimer suggesting that GSH binds to low affinity inactive enzyme dimer converting it to high affinity functionally active dimer. In order to understand the role of GSH in tetramer-dimer transition of PfGST as well as in modulation of functional activity of the enzyme, detailed structural, functional and stability studies on recombinant PfGST in the presence and absence of GSH were carried out.

Results: Our data indicate that the dimer - and not the tetramer - is the active form of PfGST, and that substrate saturation is directly paralleled by dissociation of the tetramer. Furthermore, this dissociation is a reversible process indicating that the tetramer-dimer equilibrium of PfGST is defined by the surrounding GSH concentration. Equilibrium denaturation studies show that the PfGST tetramer has significantly higher stability compared to the dimer. The enhanced stability of the tetramer is likely to be due to stronger ionic interactions existing in it.

Conclusion: This is the first report for any GST where an alteration in oligomeric structure and not just small conformational change is observed upon GSH binding to the enzyme. Furthermore we also demonstrate a reversible mechanism of regulation of functional activity of Plasmodium falciparum glutathione S-transferase via GSH induced dissociation of functionally inactive tetramer into active dimers.

Figure 1

Overexpression of PfGST in E. coli and purification of the recombinant protein over Ni-NTA agarose. A. SDS-PAGE analysis of cell lysate showing overexpression of PfGST and the purified protein. Lanes 1–4 represent molecular weight markers, supernatant of un-induced culture lysate, supernatant of induced culture lysate and purified protein, respectively. B. Molecular weight andsubunit structure of PfGST dimer and tetramer. SEC profileof (a) dimeric PfGST; obtained by incubation of protein with 2 mM GSH and run with buffer containing 2 mM GSH and (b) tetrameric PfGST; incubated and run with buffer not containing GSH on a Superdex™ 200 10/300 GL column at pH 8.0 and 25°C. The column was calibrated with standard molecular weight markers: Glucose oxidase (160 kDa), albumin (66 kDa), ovalbumin (43 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa). The curves have been displaced on Y-axis for presentation. Inset shows the SDS-PAGE profile of glutaraldehyde cross-linked PfGST protein samples. Lanes 1–4 represent molecular weight markers, uncross-linked native PfGST, glutaraldehyde cross-linked dimeric PfGST and tetrameric PfGST protein samples, respectively.

Figure 2

Effect of GSH on oligomeric status and enzymatic activity of PfGST. A. Quenching of tryptophan fluorescenceintensity and enzymatic activity of tetrameric PfGST withincreasing concentrations of GSH. In the figure, solid and hollow squares denote data for enzymatic activity and fluorescence, respectively. B. SEC profile of tetrameric PfGST incubated with increasing concentrations of GSH for 2 h at 25°C and run in the same GSH containing buffer. In the figure, a-f represent curves for tetrameric PfGST incubated with 0, 0.1, 0.2, 0.4, 0.6 and 2.0 mM GSH, respectively. The curves have been displaced on Y-axis for presentation. Inset shows the percent population of dimeric (solid squares) and tetrameric (hollow squares) species of PfGST with increasing concentration of GSH based on the data obtained from the curves displayed in the main figure.

Figure 3

Effect of GSH on oligomeric status of human GST. SEC profile of human GST. Curve "a" represent hGST incubated and run with buffer containing 2 mM GSH, while curve "b" represent hGST incubated and run with buffer not containing GSH. The curves have been displaced on Y-axis for presentation. Inset shows the SDS-PAGE profile of glutaraldehyde cross-linked hGST protein samples. Lanes 1–4 represent molecular weight markers, uncross-linked native hGST, glutaraldehyde cross-linked hGST incubated with GSH and hGST incubated without GSH protein samples, respectively.

Figure 4

Deciphering the reversibility of the tetramer-dimer transition. A. SEC profile of PfGST. Curves a-e represent tetrameric PfGST, dimeric PfGST, dimeric PfGST dialysed for 4 h in buffer devoid of GSH, dimeric PfGST dialysed for 24 h in buffer devoid of GSH, dimeric PfGST loaded on the column equilibrated and run with buffer without GSH. The curves have been displaced on Y-axis for presentation. B. Enzymatic activity assay of the eluted peaks. In the figure, bars a-e represents data for the peaks described in 4A.

Figure 5

pH-, GdnHCl- and urea- induced unfolding of dimeric and tetrameric PfGST at pH 8.0 and 25°C. A. Effects of the pH on the CD signal at 222 nm for the dimeric (solidsquares) and tetrameric (hollow squares) PfGST. The data is presented as percentage with the value observed for enzyme at pH 8.0 taken as 100%. B Effect of increasing GdnHCl concentrations on the CD ellipticity at 222 nm and the tryptophan emission wavelength maxima of dimeric and tetrameric PfGST. C. Effect of increasing urea concentrations on the CD ellipticity at 222 nm and the tryptophan emission wavelength maxima of dimeric and tetrameric PfGST. In the panel B and C, solid squares, solid circles, hollow squares and hollow circles represent data for CD of the dimers, fluorescence of the dimers, CD of the tetramers and fluorescence of the tetramers, respectively. The CD data has been presented as percentage with the value observed in the absence of denaturant (GdnHCl or urea) taken as 100%. The experimental details are mentioned in the Methods section.

Figure 6

Schematic overview of PfGST tetramer-dimer transition with respect to GSH binding, activity and stability.