Role of the Conserved DECH-Box Cysteine in Coupling Hepatitis C Virus Helicase-Catalyzed ATP Hydrolysis to RNA Unwinding.
Role of the Conserved DECH-Box Cysteine in Coupling Hepatitis C Virus Helicase-Catalyzed ATP Hydrolysis to RNA Unwinding.
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保守的 DECH-Box 半胱氨酸在丙型肝炎病毒解旋酶催化 ATP 水解与 RNA 解旋偶联中的作用。
DOI：
10.1021/acs.biochem.8b00796
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Frick,DavidN
中科院分区：
文献类型：
--
作者：
Yerukhimovich,MarkM;Marohnic,ChristopherC;Frick,DavidN
DECH盒蛋白是DExH/D-box超家族2解旋酶的一个亚类，在其ATP结合位点具有保守的Asp-Glu-Cys-His基序。保守的His帮助定位Asp和Glu残基，其协调将蛋白质连接到ATP的二价金属阳离子并激活ATP水解所需的水分子，但Cys的作用仍不清楚。本研究采用定点突变的模型DECH盒解旋酶编码的丙型肝炎病毒（HCV），以检查解旋酶的作用中的半胱氨酸。缺乏半胱氨酸的蛋白质解旋DNA的效率低于野生型蛋白质。例如，在低蛋白浓度下，含有Gly而不是DECH盒Cys的解旋酶比野生型解旋酶解旋DNA更慢，但在较高蛋白浓度下，这两种蛋白质以相似的速率解旋DNA。分析的所有HCV蛋白对ATP和核酸具有相似的亲和力，并且在RNA存在下以相似的速率水解ATP。然而，在没有RNA的情况下，所有缺乏DECH盒半胱氨酸的蛋白质水解ATP的速度比野生型蛋白质快10-15倍，Km值更高，对金属离子的表观亲和力更低。从HCV基因型2a和1b分离的蛋白质中观察到这些差异，表明这种作用是保守的。这些数据表明，解旋酶需要Cys 292在ATP不水解直到RNA结合的状态下结合ATP。
DECH-box proteins are a subset of DExH/D-box superfamily 2 helicases possessing a conserved Asp-Glu-Cys-His motif in their ATP binding site. The conserved His helps position the Asp and Glu residues, which coordinate the divalent metal cation that connects the protein to ATP and activate the water molecule needed for ATP hydrolysis, but the role of the Cys is still unclear. This study uses site-directed mutants of the model DECH-box helicase encoded by the hepatitis C virus (HCV) to examine the role of the Cys in helicase action. Proteins lacking a Cys unwound DNA less efficiently than wild-type proteins did. For example, at low protein concentrations, a helicase harboring a Gly instead of the DECH-box Cys unwound DNA more slowly than the wild-type helicase did, but at higher protein concentrations, the two proteins unwound DNA at similar rates. All HCV proteins analyzed had similar affinities for ATP and nucleic acids and hydrolyzed ATP in the presence of RNA at similar rates. However, in the absence of RNA, all proteins lacking a DECH-box cysteine hydrolyzed ATP 10–15 times faster with higherKmvalues, and lower apparent affinities for metal ions, compared to those observed with wild-type proteins. These differences were observed with proteins isolated from HCV genotypes 2a and 1b, suggesting that this role is conserved. These data suggest the helicase needs Cys292 to bind ATP in a state where ATP is not hydrolyzed until RNA binds.
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