Structural and biochemical insights into the recognition of RNA helicase CGH-1 by CAR-1 in C. elegans
Structural and biochemical insights into the recognition of RNA helicase CGH-1 by CAR-1 in C. elegans
复制标题
线虫中 CAR-1 识别 RNA 解旋酶 CGH-1 的结构和生化见解
DOI：
10.1016/j.bbrc.2021.02.119
复制
发表时间：
2021-03-03
影响因子：
3.1
通讯作者：
Hong, Jingjun
中科院分区：
文献类型：
--
作者：
Zhang, Yong;Lv, Mengqi;Hong, Jingjun
在线虫中，含有RNA解旋酶CGH-1和种系特异性RNA结合蛋白CAR-1的蛋白质-RNA复合体参与了线虫功能的各个方面。然而，这种蛋白质复合体组装的结构基础仍然不清楚。在这里，我们阐明了CAR-1识别CGH-1的分子基础。此外，我们还发现NTL1a MIF4G结构域在体外对CGH-1的ATPase活性有刺激作用。此外，我们还用X射线结晶学方法确定了CGH-1的两个类RecA结构域的结构，其分辨率分别为1.85和2.40？结构和生化方法显示CGH-1 RecA2和CAR-1的FDF-TFG基序之间存在双向界面。通过ITC和GST-Pull-down体外评估，CGH-1 RecA2或CAR-1的核磁共振和基于结构的突变减弱或破坏了CGH-1与CAR-1的结合。这些发现为CAR-1识别CGH-1的保守机制提供了见解。总之，我们的数据提供了在理解线虫中CGH-1和CAR-1的组装和功能方面缺失的物理环节。？2021爱思唯尔公司。保留所有权利。
A protein-RNA complex containing the RNA helicase CGH-1 and a germline specific RNA-binding protein CAR-1 is involved in various aspects of function in C. elegans. However, the structural basis for the assembly of this protein complex remains unclear. Here, we elucidate the molecular basis of the recognition of CGH-1 by CAR-1. Additionally, we found that the ATPase activity of CGH-1 is stimulated by NTL1a MIF4G domain in vitro. Furthermore, we determined the structures of the two RecA-like domains of CGH-1 by X-ray crystallography at resolutions of 1.85 and 2.40 ?, respectively. Structural and biochemical approaches revealed a bipartite interface between CGH-1 RecA2 and the FDF-TFG motif of CAR-1. NMR and structure-based mutations in CGH-1 RecA2 or CAR-1 attenuated or disrupted CGH-1 binding to CAR 1, assessed by ITC and GST-pulldown in vitro. These findings provide insights into a conserved mechanism in the recognition of CGH-1 by CAR-1. Together, our data provide the missing physical links in understanding the assembly and function of CGH-1 and CAR-1 in C. elegans. ? 2021 Elsevier Inc. All rights reserved.