Research Group Heussler (Malaria I) - Bernhard-Nocht-Institut für ...

Project Description and Results
A gst gene is localized on chromosome 14 of P. falciparum.
The recombinant protein is active with the model
substrate 1-chloro-2,4-dinitrobenzene, as well as cumene
hydroperoxide, exhibiting moderate non-seleniumdependent
glutathione peroxidase activity. Interestingly,
the P. falciparum protein is inhibited by hemin and ferriprotoporphyrin
IX much stronger than the human pisubclass
enzyme. Therefore it is postulated that one
function of the highly abundant protein is to sequester
nonpolymerized heme that diffuses from the food
vacuole into the cytosol and thus prevent its toxic effect
within the parasite. Inhibition of the Pf-GST is an interesting
aspect for drug design because this could intensify
the effect of drugs such as chloroquine that interfere
with heme polymerization in the food vacuole of
the parasite. On the basis of sequence similarity, gene
organisation and kinetic properties, a close relationship
of the Pf-GST to the mu- or pi-subclasses of GSTs was
observed; however, without proper knowledge of the
tertiary structure of the Pf-GST, it was not possible to
assign the enzyme to any specific subclass. To this end
we have determined the X-ray crystal structure of the
Pf-GST.
Figure 2: Ribbon diagram of (a) the Pf-GST monomer, emphasizing
the secondary structure elements. The S-hexylglutathione
molecule bound to the glutathione binding pocket is shown in
ball and stick representation. (b) The biological dimer is shown
along the 2-fold axis. The 2-fold axis is perpendicular to the
plane, and helices 3 and 4 are arranged around the 2-fold axis.
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Crystals were grown from a reservoir consisting of 2.1
M (NH4)2SO4, 0.1 M sodium cacodylate pH 6.0 and 2
mM glutathione using hanging-drop vapour diffusion
(Figure 1). The native and inhibited crystal structure of
the Pf-GST was analysed at 2.6 and 2.2 Å, respectively
(Figure 2). Pf-GST 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-hexylglutathione 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-GST 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.
Selected Publications
• Liebau E, Bergmann B, Campbell AM, Teesdale-
Spittle P, Brophy PM, Lüersen K, Walter RD. (2002).
The glutathione S-transferase from Plasmodium falciparum.
Mol Biochem Parasitol 124: 85-90
• Müller S, Liebau E, Walter RD, Krauth-Siegel RL.
(2003). Thiol-based redox metabolism of protozoan
parasites.Trends Parasitol 19:320-8
• Burmeister C, Perbandt M, Betzel C, Walter RD,
Liebau E. (2003). Crystallization and preliminary X-ray
diffraction studies of the glutathione S-transferase
from Plasmodium falciparum. Acta Crystallogr D Biol
Crystallogr 59:1469-71
• Perbandt M, Burmeister C, Walter RD, Betzel C,
Liebau E. (Epub Sep 12, 2003). Native and inhibited
structure of a Mu class-related glutathione S-transferase
from Plasmodium falciparum.
J Biol Chem 279:1336-42
Cooperating Partners
• Markus Perbandt, DESY, Hamburg
• Christian Betzel, University of Hamburg
Investigators
• Eva Liebau
• Cora Burmeister
• Rolf D. Walter
Parasitology Section