Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors.
Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors.
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DOI：
10.1074/jbc.m311744200
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发表时间：
2004-06-04
期刊：
影响因子：
--
通讯作者：
Song J
中科院分区：
文献类型：
--
作者：
Shi J;Wei Z;Song J
严重急性呼吸综合征(SARS)3C型蛋白水解酶由两个不同的结构域组成，即N端的胰凝乳酶折叠和C端的额外螺旋结构域III。以前，这个额外结构域的功能作用一直是完全未知的，人们认为冠状病毒3CL蛋白与小核糖核酸病毒3C蛋白具有相同的酶机制，后者含有胰凝乳酶折叠，但没有额外的结构域。为了了解额外结构域的功能作用，并利用动态光散射、圆二色谱和核磁共振光谱表征酶与底物的相互作用，我们1)将SARS 3CL蛋白全长切割成两个不同的折叠，并随后研究了它们的结构和二聚化性质；2)研究了三个底物多肽与整个酶及其两个解剖折叠的结构和结合作用。结果发现：1)虽然两个分离的部分折叠成天然的类结构，但凝乳酶折叠的活性与整个酶相比很弱；2)尽管凝乳酶折叠在很大的蛋白质浓度范围内仍然是单体，但额外的结构域即使在很低的浓度下也以稳定的二聚体存在。这一观察结果有力地表明，额外的结构域有助于SARS 3CL蛋白酶的二聚化，从而将酶从失活形式(单体)切换到活性形式(二聚体)。这一发现不仅从酶机制上分离了冠状病毒3CL蛋白水解酶和微小冠状病毒3C蛋白水解酶，而且还将额外螺旋结构域上的二聚化界面定义为设计特异性蛋白水解酶抑制剂的新靶点。此外，底物肽S1的首选溶液构象的确定以及核磁共振差示线宽和转移核Overhauser增强研究使我们能够精确定位S1肽的结合结构。
The severe acute respiratory syndrome (SARS) 3C-like protease consists of two distinct folds, namely the N-terminal chymotrypsin fold containing the domains I and II hosting the complete catalytic machinery and the C-terminal extra helical domain III unique for the coronavirus 3CL proteases. Previously the functional role of this extra domain has been completely unknown, and it was believed that the coronavirus 3CL proteases share the same enzymatic mechanism with picornavirus 3C proteases, which contain the chymotrypsin fold but have no extra domain. To understand the functional role of the extra domain and to characterize the enzyme-substrate interactions by use of the dynamic light scattering, circular dichroism, and NMR spectroscopy, we 1) dissected the full-length SARS 3CL protease into two distinct folds and subsequently investigated their structural and dimerization properties and 2) studied the structural and binding interactions of three substrate peptides with the entire enzyme and its two dissected folds. The results lead to several findings; 1) although two dissected parts folded into the native-like structures, the chymotrypsin fold only had weak activity as compared with the entire enzyme, and 2) although the chymotrypsin fold remained a monomer within a wide range of protein concentrations, the extra domain existed as a stable dimer even at a very low concentration. This observation strongly indicates that the extra domain contributes to the dimerization of the SARS 3CL protease, thus, switching the enzyme from the inactive form (monomer) to the active form (dimer). This discovery not only separates the coronavirus 3CL protease from the picornavirus 3C protease in terms of the enzymatic mechanism but also defines the dimerization interface on the extra helical domain as a new target for design of the specific protease inhibitors. Furthermore, the determination of the preferred solution conformation of the substrate peptide S1 together with the NMR differential line-broadening and transferred nuclear Overhauser enhancement study allows us to pinpoint the bound structure of the S1 peptide.