Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity
Poliovirus 2C Protein Forms Homo-oligomeric Structures Required for ATPase Activity
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DOI：
10.1074/jbc.m109.031807
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发表时间：
2009-08-14
影响因子：
4.8
通讯作者：
Ehrenfeld, Ellie
中科院分区：
文献类型：
--
作者：
Adams, Peter;Kandiah, Eaazhisai;Ehrenfeld, Ellie
脊髓灰质炎病毒蛋白2C在病毒RNA复制中起重要作用，尽管其精确的生物化学活性或结构要求尚未阐明。该蛋白具有几种独特的性质，包括ATP酶活性和膜与RNA结合，这些性质在许多正链RNA病毒的直系同源物中是保守的。序列比对将这些蛋白质置于SF 3解旋酶家族中，其为AAA+ ATP酶超家族的子集。AAA+蛋白的一个共同特征是形成对其催化功能至关重要的寡聚环。在这里，我们表明，重组蛋白，MBP-2C，其中麦芽糖结合蛋白融合到2C，形成可溶性低聚物和ATP酶活性被限制在低聚物含有馏分从凝胶过滤色谱。通过负染色电子显微镜观察，活性部分为由5-8个原聚体组成的环状颗粒。通过扫描透射电子显微镜获得的质量测量证实了这一结论。在2C核苷酸结合域的氨基酸残基的突变表明，结合或水解ATP的能力的损失不影响寡聚化。活性MBP-2C和失活突变蛋白的共表达产生了混合的寡聚体，其表现出很小的ATP酶活性，这表明掺入失活亚基消除了整个颗粒的功能。最后，N-末端38个氨基酸的缺失阻断了融合蛋白的寡聚化并消除了ATP酶活性，尽管保留了未改变的核苷酸结合结构域。
The poliovirus protein 2C plays an essential role in viral RNA replication, although its precise biochemical activities or structural requirements have not been elucidated. The protein has several distinctive properties, including ATPase activity and membrane and RNA binding, that are conserved among orthologs of many positive-strand RNA viruses. Sequence alignments have placed these proteins in the SF3 helicase family, a subset of the AAA+ ATPase superfamily. A feature common to AAA+ proteins is the formation of oligomeric rings that are essential for their catalytic functions. Here we show that a recombinant protein, MBP-2C, in which maltose-binding protein was fused to 2C, formed soluble oligomers and that ATPase activity was restricted to oligomer-containing fractions from gel-filtration chromatography. The active fraction was visualized by negative-staining electron microscopy as ring-like particles composed of 5-8 protomers. This conclusion was confirmed by mass measurements obtained by scanning transmission electron microscopy. Mutation of amino acid residues in the 2C nucleotide-binding domain demonstrated that loss of the ability to bind or hydrolyze ATP did not affect oligomerization. Co-expression of active MBP-2C and inactive mutant proteins generated mixed oligomers that exhibited little ATPase activity, suggesting that incorporation of inactive subunits eliminates the function of the entire particle. Finally, deletion of the N-terminal 38 amino acids blocked oligomerization of the fusion protein and eliminated ATPase activity, despite retention of an unaltered nucleotide-binding domain.