Tetramerization and cooperativity in Plasmodium falciparum glutathione S-transferase are mediated by atypic loop 113-119.
Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113-119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.
Liebau
Eva
E
Institute of Animal Physiology, University of Münster, Hindenburgplatz, 55 Münster, Germany.
Dawood
Kutayba F
KF
the Departments of Chemical Sciences and Technologies, 00133 Rome, Italy.
Fabrini
Raffaele
R
the Departments of Chemical Sciences and Technologies, 00133 Rome, Italy.
Fischer-Riepe
Lena
L
Institute of Animal Physiology, University of Münster, Hindenburgplatz, 55 Münster, Germany.
Perbandt
Markus
M
Institute of Biochemistry, Center for Structural and Cell Biology, University of Luebeck, Ratzeburger Allee 160, 23538 Luebeck, Germany; Laboratory for Structural Biology of Infection and Inflammation, Deutsches Elektronen Synchrotron, Notkestrasse 85, 22603 Hamburg, Germany.
Stella
Lorenzo
L
the Departments of Chemical Sciences and Technologies, 00133 Rome, Italy.
Pedersen
Jens Z
JZ
Biology, University of Rome "Tor Vergata," 00133 Rome, Italy.
Bocedi
Alessio
A
Department of Biology, University of Rome "Roma Tre," 00146 Rome, Italy; Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina 27708.
Petrarca
Patrizia
P
Biology, University of Rome "Tor Vergata," 00133 Rome, Italy.
Federici
Giorgio
G
Childrens Hospital "Bambin Gesù," 00165 Rome, Italy.
Ricci
Giorgio
G
the Departments of Chemical Sciences and Technologies, 00133 Rome, Italy.
D000818:Animals; D001665:Binding Sites; D019281:Dimerization; D005978:Glutathione / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism*; D006427:Hemin / Q000737:chemistry; D007700:Kinetics; D008954:Models, Biological; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010963:Plasmodium falciparum / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
D006427:Hemin; D005982:Glutathione Transferase; D005978:Glutathione