Alteration of molecular assembly of peroxiredoxins from hyperthermophilic archaea
Alteration of molecular assembly of peroxiredoxins from hyperthermophilic archaea
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超嗜热古菌过氧化还原蛋白分子组装的改变
DOI：
10.1093/jb/mvx045
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Uegaki Koichi
中科院分区：
文献类型：
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作者：
Nakamura Tsutomu;Oshima Maki;Yasuda Megumi;Shimamura Akiko;Morita Junji;Uegaki Koichi
来自堀越火球菌的过氧化还原蛋白（PhPrx）是由五个二聚体环型组装形成的十聚体蛋白质。为了设计 PhPrx 的四级结构，我们通过引入旨在将 PhPrx 解离成二聚体的六个点突变来创建突变体 PhPrx (PhPrx6m)。尽管 PhPrx6m 在溶液中是二聚体，但在结晶后这六个二聚体组装成十二聚体。在晶体结构中，PhPrx6m被过氧化，过氧化的半胱氨酸呈磺酸形式，C端区域的两个半胱氨酸通过分子内二硫键连接。因此，我们表征了过氧化氢过氧化的野生型 PhPrx (PhPrxPer)。分析超速离心表明 PhPrxPer 在溶液中的分子量高于 PhPrx。通过分析 PhPrxPer 的晶体结构证实了这一点，发现 PhPrxPer 形成由六个二聚体组成的环状十二聚体。 PhPrx6m 和 PhPrxPer 的单体结构与 PhPrx 的单体结构在两个结构域的相对方向上有所不同，反映了环型组装中二聚体的数量。与 PhPrx 不同，来自 Pernix 的同源过氧化还原蛋白 (ApPrx) 不发生六聚体缔合。这一特性可以通过 ApPrx 中两个结构域之间更强的连接来解释，因为 ApPrx 的 C 端延伸相对于 PhPrx。
Peroxiredoxin fromPyrococcus horikoshii(PhPrx) is a decameric protein formed by ring-type assembly of five dimers. To engineer the quaternary structure of PhPrx, we created a mutant PhPrx (PhPrx6m) by introducing six point mutations designed to dissociate PhPrx into dimers. Although PhPrx6m was a dimer in solution, the six dimers assembled into a dodecamer following crystallization. In the crystal structure, PhPrx6m was overoxidized, and the peroxidatic cysteine was in sulfonic acid form and two cysteines in the C-terminal region were linked by an intramolecular disulfide bond. Thus, we characterized the wild-type PhPrx overoxidized by hydrogen peroxide (PhPrxPer). Analytical ultracentrifugation showed that PhPrxPer had a higher molecular mass in solution than PhPrx. This was confirmed by analysis of the crystal structure of PhPrxPer, which was found to form a ring-type dodecamer composed of six dimers. The monomeric structures of PhPrx6m and PhPrxPer differed from that of PhPrx in the relative orientation of two domains, reflecting the number of dimers in the ring-type assembly. Unlike PhPrx, homologous peroxiredoxin fromAeropyrum pernix(ApPrx) did not undergo hexameric association. This property can be explained by the stronger connection between the two domains in ApPrx due to its C-terminal extension relative to PhPrx.