Sorrentino Luca, Calogero Alessandra Maria, Pandini Vittorio, Vanoni Maria Antonietta, Sevrioukova Irina F, Aliverti Alessandro
Department of Biosciences, Università degli Studi di Milano , via Celoria 26, 20133 Milano, Italy.
Department of Molecular Biology and Biochemistry, University of California , Irvine, California 92697-3900, United States.
Biochemistry. 2015 Dec 1;54(47):6996-7009. doi: 10.1021/acs.biochem.5b00898. Epub 2015 Nov 16.
Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.
凋亡诱导因子（AIF）是一种具有促生存和促死亡活性的线粒体黄素蛋白，在线粒体能量代谢和不依赖半胱天冬酶的凋亡中起关键作用。AIF结构或表达的缺陷可导致线粒体异常，进而引起线粒体缺陷和神经退行性变。虽然对AIF诱导凋亡的机制进行了广泛研究，但对其线粒体功能却知之甚少。AIF的一个独特特征是，它在与NADH反应时能够形成紧密的、对空气稳定的电荷转移（CT）复合物，并经历构象转换导致二聚化，这一过程被认为对其重要和致命功能至关重要。尽管已经分析了AIF与NAD(+)/H相互作用的某些方面，但其精确机制尚未完全了解。我们研究了小鼠AIF的氧化型和光还原型野生型以及G307A和 -E变体如何与NAD(+)/H和烟酰胺单核苷酸（NMN(+)/H）结合，以确定腺苷酸部分在结合过程中的作用。我们的结果表明：（i）NAD(+)/H的腺苷酸部分对于与AIF的结合以及复合物随后的结构重组至关重要，但对蛋白质二聚化并非如此；（ii）FAD的还原而非NAD(+)/H与AIF的结合启动构象重排；（iii）G307E（相当于人类AIF中的病理性G308E突变）或G307A取代导致的腺苷酸结合位点改变会降低NAD(+)/H的亲和力和结合速率，进而扰乱CT复合物的形成和蛋白质二聚化，但对调节肽中的构象转换没有影响。
