Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies
Insights to substrate binding and processing by West Nile Virus NS3 protease through combined modeling, protease mutagenesis, and kinetic studies
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DOI：
10.1074/jbc.m607641200
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发表时间：
2006-12-15
影响因子：
4.8
通讯作者：
Young, Paul R.
中科院分区：
文献类型：
--
作者：
Chappell, Keith J.;Stoermer, Martin J.;Young, Paul R.
西尼罗河病毒正在成为一种广泛传播的病原体，感染至少四大洲的人们，而这些感染或其许多相关病症却没有有效的治疗方法。病毒复制所必需的关键酶是病毒蛋白酶 NS2B-NS3，它在所有黄病毒中高度保守。通过结合底物与 NS3 晶体结构活性位点的分子拟合、定点酶和辅因子诱变以及短肽底物组蛋白水解加工的动力学研究，我们已经确定了定义 NS3 蛋白酶底物特异性的重要酶-底物相互作用。除了更好地了解 S2、S3 和 S4 酶残基在底物结合中的参与之外，还发现辅因子 NS2B 内的残基会强烈影响黄病毒蛋白酶对底物 P2 处赖氨酸或精氨酸的偏好。四肽底物的优化以增强蛋白酶亲和力和加工效率也为开发西尼罗病毒感染抑制剂提供了重要线索。
West Nile Virus is becoming a widespread pathogen, infecting people on at least four continents with no effective treatment for these infections or many of their associated pathologies. A key enzyme that is essential for viral replication is the viral protease NS2B-NS3, which is highly conserved among all flaviviruses. Using a combination of molecular fitting of substrates to the active site of the crystal structure of NS3,site-directed enzyme and cofactor mutagenesis, and kinetic studies on proteolytic processing of panels of short peptide substrates, we have identified important enzyme-substrate interactions that define substrate specificity for NS3 protease. In addition to better understanding the involvement of S2, S3, and S4 enzyme residues in substrate binding, a residue within cofactor NS2B has been found to strongly influence the preference of flavivirus proteases for lysine or arginine at P2 in substrates. Optimization of tetrapeptide substrates for enhanced protease affinity and processing efficiency has also provided important clues for developing inhibitors of West Nile Virus infection.