Singh Kulwant, Bhakuni Vinod
Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow-226 001, India.
Proteins. 2008 Jun;71(4):1879-88. doi: 10.1002/prot.21872.
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.
顶质体及其所含蛋白质是顶复门寄生虫化疗的特异性靶点。铁氧化还原蛋白-NADP(+)还原酶（FNR）是刚地弓形虫顶质体中存在的一种重要酶，在许多不同电子传递途径的分支步骤中作为通用电子开关发挥作用。尽管它作为药物靶点很重要，但关于该酶的结构信息却很少。我们结合荧光光谱、圆二色光谱、酶活性测定和尺寸排阻色谱，研究了重组刚地弓形虫铁氧化还原蛋白-NADP(+)还原酶（TgFNR）结构和功能特性的pH依赖性变化以及结构域间相互作用，以了解负责稳定天然构象和调节酶功能活性的相互作用。在生理条件下，重组TgFNR以开放构象稳定存在。发现该酶的开放构象对其最佳功能至关重要，因为通过调节pH诱导紧密性/刚性会导致功能活性降低。在天然构象中，NADP(+)结合结构域和FAD结合结构域之间存在强烈相互作用，从而使该酶成为一种结构协同分子。在酸性条件下（pH约为4），天然TgFNR中存在的结构域间相互作用丧失，该酶在结构上变得不协同。pH诱导的NADP(+)结合结构域结构改变，更确切地说是构象的紧密化，导致其对热变性稳定。这些研究证明了静电相互作用在稳定TgFNR天然构象和维持结构协同性方面的重要性。
