Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron−sulfur clusters of human mitoNEET.
Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron−sulfur clusters of human mitoNEET.
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DOI：
10.1021/bi101168c
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发表时间：
2010-10
期刊：
影响因子：
2.9
通讯作者：
Tao Zhou;Jinzhong Lin;Yingang Feng;Jinfeng Wang
中科院分区：
文献类型：
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作者：
Tao Zhou;Jinzhong Lin;Yingang Feng;Jinfeng Wang
线粒体外膜蛋白mitoNEET是抗糖尿病药物吡格列酮的细胞靶点。与吡格列酮结合稳定蛋白，防止[2Fe-2S]簇释放。在这里，我们报道了还原性烟酰胺腺嘌呤二核苷酸磷酸（NADPH）可以与同二聚体mitoNEET结合，影响每个亚基在环区（Y71−H87）内结合的[2Fe-2S]簇的稳定性。核磁共振（NMR）和等温滴定量热实验表明，NADPH与mitoNEET（44−108）结合较弱，mitoNEET是mitoNEET的可溶性结构域，含有44−108残基。可见-紫外吸收测量揭示了NADP结合对[2Fe-2S]簇的不稳定作用。通过核磁共振和圆二色性实验观察到铁-硫簇分解导致mitoNEET（44−108）的三维结构被破坏。在pH≤7.0时，NADPH的结合促进了铁-硫簇从蛋白质中释放。mitoNEET每个亚基的残基K55和H58被证明参与NADPH的结合。NADPH结合可能干扰来自一个亚基的K55和H58分别与来自另一个亚基的H87 ‘和R73 ’的相互作用，从而干扰[2Fe-2S]簇结合。这可能解释了NADPH结合在[2Fe-2S]簇上的不稳定效应。
The outer mitochondrial membrane protein mitoNEET is a cellular target of the antidiabetic drug pioglitazone. Binding of pioglitazone stabilizes the protein against [2Fe-2S] cluster release. Here, we report that reduced nicotinamide adenine dinucleotide phosphate (NADPH) can bind to homodimeric mitoNEET, influencing the stability of the [2Fe-2S] cluster that is bound within a loop region (Y71−H87) in each subunit. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry experiments demonstrated that NADPH binds weakly to mitoNEET(44−108), a soluble domain of mitoNEET containing residues 44−108. Visible−UV absorption measurements revealed the destabilizing effect of NADP binding on the [2Fe-2S] clusters. Disruption of the three-dimensional structure of mitoNEET(44−108) as a result of decomposition of the iron−sulfur clusters was observed by NMR and circular dichroism experiments. Binding of NADPH facilitated release of the iron−sulfur clusters from the protein at pH≤7.0. Residues K55 and H58 of each subunit of mitoNEET were shown to be involved in NADPH binding. NADPH binding may perturb the interactions of K55 and H58 from one subunit with H87′ and R73′, respectively, from the other subunit, thereby interfering with [2Fe-2S] cluster binding. This may account for the destabilization effect of NADPH binding on the [2Fe-2S] clusters.