Department of Structural Chemistry, Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, USA.
J Mol Biol. 2011 Feb 4;405(5):1139-53. doi: 10.1016/j.jmb.2010.11.034. Epub 2010 Dec 9.
The structural mechanism by which nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) translocates along RNA is currently unknown. HCV NS3 is an ATP-dependent motor protein essential for viral replication and a member of the superfamily 2 helicases. Crystallographic analysis using a labeled RNA oligonucleotide allowed us to unambiguously track the positional changes of RNA bound to full-length HCV NS3 during two discrete steps of the ATP hydrolytic cycle. The crystal structures of HCV NS3, NS3 bound to bromine-labeled RNA, and a tertiary complex of NS3 bound to labeled RNA and a non-hydrolyzable ATP analog provide a direct view of how large domain movements resulting from ATP binding and hydrolysis allow the enzyme to translocate along the phosphodiester backbone. While directional translocation of HCV NS3 by a single base pair per ATP hydrolyzed is observed, the 3' end of the RNA does not shift register with respect to a conserved tryptophan residue, supporting a "spring-loading" mechanism that leads to larger steps by the enzyme as it moves along a nucleic acid substrate.
丙型肝炎病毒（HCV）非结构蛋白 3（NS3）沿 RNA 转移的结构机制目前尚不清楚。HCV NS3 是一种依赖于 ATP 的运动蛋白，是病毒复制所必需的，也是超家族 2 解旋酶的成员。使用标记的 RNA 寡核苷酸进行晶体学分析，使我们能够在 ATP 水解循环的两个离散步骤中明确跟踪与全长 HCV NS3 结合的 RNA 的位置变化。HCV NS3、与溴标记的 RNA 结合的 NS3 以及与标记的 RNA 和非水解型 ATP 类似物结合的 NS3 的三级复合物提供了一个直接的视角，了解 ATP 结合和水解导致的大结构域运动如何使酶沿磷酸二酯骨架转移。虽然观察到 HCV NS3 每水解一个 ATP 就会沿单个碱基对进行定向转移，但 RNA 的 3' 末端相对于保守的色氨酸残基不会改变登记，这支持了一种“弹簧加载”机制，当酶沿核酸底物移动时，该机制会导致更大的步长。
