Structural basis for shieldin complex subunit 3?mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair
Structural basis for shieldin complex subunit 3?mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair
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屏蔽蛋白复合物亚基 3 的结构基础？在 DNA 双链断裂修复过程中介导检查点蛋白 REV7 的募集
DOI：
10.1074/jbc.ra119.011464
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发表时间：
2020-01-03
影响因子：
4.8
通讯作者：
Zhou, Zheng
中科院分区：
文献类型：
--
作者：
Dai, Yaxin;Zhang, Fan;Zhou, Zheng
shieldin complex subunit 3（SHLD 3）是最近鉴定的shieldin complex的顶端亚基，在DNA双链断裂修复中起关键作用。为了实现其在DNA修复中的功能，SHLD3与有丝分裂纺锤体组装检查点蛋白REV7同源物（REV7）相互作用，但这种相互作用的细节仍然不清楚。在这里，我们提出的晶体结构的REV7在复杂的SHLD3的REV7结合域（RBD）在2.2？2.3?决议。结构显示SHLD3中的勺形RBD使用其N-末端环和C-末端？helix（？C-螺旋）与REV7相互作用。的N-末端环表现出类似的结构，以前确定的其他REV7结合蛋白，和保守性较低？C-螺旋区与REV7的结合方式不同。在体外和体内结合分析表明，N-末端环和？C-螺旋都是不可缺少的高亲和力REV7结合（低纳摩尔亲和力），强调SHLD3的关键作用？蛋白质结合中的C螺旋。此外，结合动力学分析表明，REV7？安全带？区域，其中发挥作用，在结合其他蛋白质，是必不可少的SHLD3？REV7结合，因为该区域延迟RBD从结合的REV7解离。总之，我们的研究结果揭示了SHLD3？REV7交互，并提供SHLD3如何识别REV7的关键见解。
Shieldin complex subunit 3 (SHLD3) is the apical subunit of a recently-identified shieldin complex and plays a critical role in DNA double-strand break repair. To fulfill its function in DNA repair, SHLD3 interacts with the mitotic spindle assembly checkpoint protein REV7 homolog (REV7), but the details of this interaction remain obscure. Here, we present the crystal structures of REV7 in complex with SHLD3's REV7-binding domain (RBD) at 2.2?2.3 ? resolutions. The structures revealed that the ladle-shaped RBD in SHLD3 uses its N-terminal loop and C-terminal ?-helix (?C-helix) in its interaction with REV7. The N-terminal loop exhibited a structure similar to those previously identified in other REV7-binding proteins, and the less-conserved ?C-helix region adopted a distinct mode for binding REV7. In vitro and in vivo binding analyses revealed that the N-terminal loop and the ?C-helix are both indispensable for high-affinity REV7 binding (with low-nanomolar affinity), underscoring the crucial role of SHLD3 ?C-helix in protein binding. Moreover, binding kinetics analyses revealed that the REV7 ?safety belt? region, which plays a role in binding other proteins, is essential for SHLD3?REV7 binding, as this region retards the dissociation of the RBD from the bound REV7. Together, the findings of our study reveal the molecular basis of the SHLD3?REV7 interaction and provide critical insights into how SHLD3 recognizes REV7.