Maturation mechanism of severe acute respiratory syndrome (SARS) coronavirus 3C-like proteinase.
Maturation mechanism of severe acute respiratory syndrome (SARS) coronavirus 3C-like proteinase.
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DOI：
10.1074/jbc.m109.095851
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发表时间：
2010-09-03
期刊：
影响因子：
--
通讯作者：
Lai L
中科院分区：
文献类型：
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作者：
Li C;Qi Y;Teng X;Yang Z;Wei P;Zhang C;Tan L;Zhou L;Liu Y;Lai L
严重急性呼吸综合征（SARS）冠状病毒的3C样蛋白酶（3CLpro）在病毒成熟过程中起着重要作用，被认为是抗SARS药物设计的重要靶点。各种体外研究表明，只有成熟的3CLpro的二聚体是有活性的。然而，由于内部编码的3CLpro通过在N-末端和C-末端侧翼位点处的自溶切割从复制酶多聚蛋白成熟，因此尚不清楚多聚蛋白是否也需要首先二聚化以进行其自切割反应。我们构建了一个包含青色荧光蛋白（C）、SARS 3CLpro（XX）N端侧翼底物肽、SARS 3CLpro（3CLP）和黄色荧光蛋白（Y）的大蛋白，以利用荧光共振能量转移研究3CLpro的自加工。与成熟的3CLpro相比，多聚蛋白以及一步消化产物3CLP-Y-His在凝胶过滤和分析超滤分析中显示为单体。然而，当在化学交联实验和分析超离心分析中将这些大蛋白与底物类似物化合物一起孵育时，仍然可以诱导和检测到二聚体。我们还测量了不同酶浓度下的酶活性，发现底物诱导二聚体形成的明显趋势。基于这些发现，我们得出结论，底物诱导的二聚化是必不可少的活性SARS-3CLpro的多蛋白，并提出了一个修改后的模型3CLpro成熟过程。由于许多病毒蛋白酶经历类似的成熟过程，该模型可能普遍适用。
The 3C-like proteinase (3CLpro) of the severe acute respiratory syndrome (SARS) coronavirus plays a vital role in virus maturation and is proposed to be a key target for drug design against SARS. Various in vitro studies revealed that only the dimer of the matured 3CLpro is active. However, as the internally encoded 3CLpro gets matured from the replicase polyprotein by autolytic cleavage at both the N-terminal and the C-terminal flanking sites, it is unclear whether the polyprotein also needs to dimerize first for its autocleavage reaction. We constructed a large protein containing the cyan fluorescent protein (C), the N-terminal flanking substrate peptide of SARS 3CLpro (XX), SARS 3CLpro (3CLP), and the yellow fluorescent protein (Y) to study the autoprocessing of 3CLpro using fluorescence resonance energy transfer. In contrast to the matured 3CLpro, the polyprotein, as well as the one-step digested product, 3CLP-Y-His, were shown to be monomeric in gel filtration and analytic ultracentrifuge analysis. However, dimers can still be induced and detected when incubating these large proteins with a substrate analog compound in both chemical cross-linking experiments and analytic ultracentrifuge analysis. We also measured enzyme activity under different enzyme concentrations and found a clear tendency of substrate-induced dimer formation. Based on these discoveries, we conclude that substrate-induced dimerization is essential for the activity of SARS-3CLpro in the polyprotein, and a modified model for the 3CLpro maturation process was proposed. As many viral proteases undergo a similar maturation process, this model might be generally applicable.