Department of Chemistry & Biochemistry , University of Wisconsin-Milwaukee , Milwaukee , Wisconsin 53211 , United States.
Abbott Laboratories , 100 Abbott Park Road , Abbott Park , Illinois 60064 , United States.
Biochemistry. 2018 Oct 30;57(43):6247-6255. doi: 10.1021/acs.biochem.8b00796. Epub 2018 Oct 16.
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 higher K values, 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.
DECH-box 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 有助于定位 Asp 和 Glu 残基，它们与连接蛋白和 ATP 的二价金属阳离子配位，并激活水解 ATP 所需的水分子，但 Cys 的作用仍不清楚。本研究使用丙型肝炎病毒 (HCV) 编码的模型 DECH-box 解旋酶的定点突变体来研究 Cys 在解旋酶作用中的作用。缺乏 Cys 的蛋白比野生型蛋白更有效地解开 DNA。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开 DNA 的速度更慢，但在更高的蛋白浓度下，两种蛋白以相似的速度解开 DNA。所有分析的 HCV 蛋白对 ATP 和核酸具有相似的亲和力，并在 RNA 存在下以相似的速度水解 ATP。然而，在没有 RNA 的情况下，所有缺乏 DECH 盒半胱氨酸的蛋白以更高的 K 值，以及更低的金属离子表观亲和力，比野生型蛋白更快地水解 ATP10-15 倍。这些差异在从 HCV 基因型 2a 和 1b 分离的蛋白中观察到，表明该作用是保守的。这些数据表明，解旋酶需要 Cys292 来结合处于不水解 ATP 的状态，直到 RNA 结合。
