Mechanism of activation of the human cysteine desulfurase complex by frataxin
Mechanism of activation of the human cysteine desulfurase complex by frataxin
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DOI：
10.1073/pnas.1909535116
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发表时间：
2019-09-24
影响因子：
11.1
通讯作者：
Barondeau, David P.
中科院分区：
文献类型：
--
作者：
Patra, Shachin;Barondeau, David P.
frataxin (FXN) 的功能引起了极大的科学兴趣，因为它的消耗与无法治愈的神经退行性疾病弗里德赖希共济失调 (FRDA) 有关。 FXN 已被证明是铁硫 (Fe-S) 簇生物合成和正常线粒体所必需的！功能。 Fe-S簇组装复合物的结构和功能核心是低活性吡哆醛5'-磷酸（PLP）依赖性半胱氨酸脱硫酶，由催化（NFS1）、LYRM蛋白（ISD11）和酰基载体蛋白（ACP）亚基组成。尽管之前的研究表明 FXN 会刺激这种组装复合物的活性，但人们对 FXN 激活的机制知之甚少。在这里，我们开发了一种放射性标记测定法，并使用停流动力学来确定 FXN 在功能上与 NFS1 的移动 S 转移环半胱氨酸相关。我们的结果支持了这个必需的半胱氨酸残基在底物结合中的关键作用，作为促进Cys-醌类PLP中间体的通用酸，作为形成NFS1过硫化物的亲核试剂，以及作为在Fe-S支架蛋白ISCU2上生成过硫化物物质的硫输送剂。 FXN 特别加速了该机制中的每个单独步骤。我们所得的结构开关模型解释了为什么人类 Fe-S 组装系统具有较低的固有活性并需要激活、功能性移动 S 转移环半胱氨酸和 FXN 结合之间的连接，以及为什么原核系统不需要类似的基于 FXN 的激活。总之，这些结果为 FXN 的变构激活剂作用提供了机制见解，并提出了在 FRDA 治疗中替代 FXN 功能的新策略。
The function of frataxin (FXN) has garnered great scientific interest since its depletion was linked to the incurable neurodegenerative disease Friedreich's ataxia (FRDA). FXN has been shown to be necessary for iron-sulfur (Fe-S) cluster biosynthesis and proper mitochondria! function. The structural and functional core of the Fe-S cluster assembly complex is a low-activity pyridoxal 5'-phosphate (PLP)-dependent cysteine desulfurase enzyme that consists of catalytic (NFS1), LYRM protein (ISD11), and acyl carrier protein (ACP) subunits. Although previous studies show that FXN stimulates the activity of this assembly complex, the mechanism of FXN activation is poorly understood. Here, we develop a radiolabeling assay and use stopped-flow kinetics to establish that FXN is functionally linked to the mobile S-transfer loop cysteine of NFS1. Our results support key roles for this essential cysteine residue in substrate binding, as a general acid to advance the Cys-quinonoid PLP intermediate, as a nucleophile to form an NFS1 persulfide, and as a sulfur delivery agent to generate a persulfide species on the Fe-S scaffold protein ISCU2. FXN specifically accelerates each of these individual steps in the mechanism. Our resulting architectural switch model explains why the human Fe-S assembly system has low inherent activity and requires activation, the connection between the functional mobile S-transfer loop cysteine and FXN binding, and why the prokaryotic system does not require a similar FXN-based activation. Together, these results provide mechanistic insights into the allosteric-activator role of FXN and suggest new strategies to replace FXN function in the treatment of FRDA.