Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode.
Liberation of SARS-CoV main protease from the viral polyprotein: N-terminal autocleavage does not depend on the mature dimerization mode.
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DOI：
10.1007/s13238-010-0011-4
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发表时间：
2010-01
期刊：
影响因子：
21.1
通讯作者：
Hilgenfeld R
中科院分区：
文献类型：
--
作者：
Chen S;Jonas F;Shen C;Hilgenfeld R
在SARS冠状病毒(SARS-CoV)复制周期中，主要蛋白水解酶(MPRO)在蛋白质的降解过程中起着至关重要的作用。这种酶的二聚化已被证明是反式切割活性不可缺少的。然而，MPRO的自动处理机制，即它自己通过自动切割从多蛋白中释放出来的机制，仍然不清楚。本研究阐明了N端自切割活性与“未成熟”MPRO二聚化的关系。选择三个残基(Arg4、Glu290和Arg298)对成熟MPRO的活性二聚体构象进行突变分析。令人惊讶的是，这三个突变体都仍然执行N端自切割，而成熟蛋白酶的二聚化和自动加工后的跨切割活性完全被E290R和R298E突变抑制，而R4E突变部分抑制。此外，成熟的E290R突变体与未成熟的C145A/E290R双突变体混合时，可以恢复N端的自切割活性，但其反式切割活性仍然缺失。因此，MPRO的N-末端自动加工似乎只需要两个相互靠近的“未成熟”单体来形成一个“中间”二聚体结构，而不是严格依赖于成熟蛋白酶中存在的活性二聚体构象。综上所述，提出了MPRO从多蛋白中自动释放的模型，这将有助于理解MPRO的自动处理机制以及SARS-CoV MPRO的自切和反切蛋白分解活性的差异。
The main protease (Mpro) plays a vital role in proteolytic processing of the polyproteins in the replicative cycle of SARS coronavirus (SARS-CoV). Dimerization of this enzyme has been shown to be indispensable for transcleavage activity. However, the auto-processing mechanism of Mpro, i.e. its own release from the polyproteins through autocleavage, remains unclear. This study elucidates the relationship between the N-terminal autocleavage activity and the dimerization of “immature” Mpro. Three residues (Arg4, Glu290, and Arg298), which contribute to the active dimer conformation of mature Mpro, are selected for mutational analyses. Surprisingly, all three mutants still perform N-terminal autocleavage, while the dimerization of mature protease and transcleavage activity following auto-processing are completely inhibited by the E290R and R298E mutations and partially so by the R4E mutation. Furthermore, the mature E290R mutant can resume N-terminal autocleavage activity when mixed with the “immature” C145A/E290R double mutant whereas its trans-cleavage activity remains absent. Therefore, the N-terminal auto-processing of Mpro appears to require only two “immature” monomers approaching one another to form an “intermediate” dimer structure and does not strictly depend on the active dimer conformation existing in mature protease. In conclusion, an auto-release model of Mpro from the polyproteins is proposed, which will help understand the auto-processing mechanism and the difference between the autocleavage and trans-cleavage proteolytic activities of SARS-CoV Mpro.