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Toxoplasma gondii ferredoxin‐NADP + reductase: Role of ionic interactions in stabilization of native conformation and structural cooperativity
Author(s) -
Singh Kulwant,
Bhakuni Vinod
Publication year - 2008
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21872
Subject(s) - ferredoxin , cooperativity , oxidoreductase , cooperative binding , ferredoxin—nadp(+) reductase , enzyme , native state , circular dichroism , electron transfer , protein structure , chemistry , biophysics , biochemistry , biology , organic chemistry
The apicoplast and the proteins present therein are parasite‐specific targets for chemotherapy of apicomplexan parasites. Ferredoxin‐NADP + reductase (FNR) is an important enzyme present in the apicoplast of Toxoplasma gondii that operates as a general electron switch at the bifurcation step of many different electron transfer pathways. In spite of its importance as drug target not much structural information on the enzyme is available. Using fluorescence and CD spectroscopy in combination with enzyme activity measurement and size exclusion chromatography, we studied the pH‐dependent changes in structural and functional properties and interdomain interactions in recombinant Toxoplasma gondii ferredoxin‐NADP + reductase (TgFNR) to understand the interactions responsible for stabilization of native conformation and modulation of functional activity of the enzyme. Under physiological conditions, the recombinant TgFNR is stabilized in an open conformation. The open conformation of the enzyme was found to be essential for its optimum functioning, as induction of compactness/rigidity by modulation of pH, leads to decrease in the functional activity. In native conformation, strong interactions exist between the NADP + ‐ and FAD‐binding domains thus making the enzyme a structurally cooperative molecule. Under acidic conditions (pH about 4), the interdomain interactions present in native TgFNR were lost and the enzyme became structurally noncooperative. The pH‐induced structural alterations in the NADP + binding domain, more precisely compaction of the conformation lead to its stabilization against thermal denaturation. The studies demonstrate the significance of electrostatic interactions both in stabilization of native conformation and maintenance of structural cooperativity in TgFNR. Proteins 2008. © 2008 Wiley‐Liss, Inc.