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首页> 外文期刊>Biochemistry >Molecular Determinants of Polyubiquitin Recognition by Continuous Ubiquitin-Binding Domains of Rad18
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Molecular Determinants of Polyubiquitin Recognition by Continuous Ubiquitin-Binding Domains of Rad18

机译：Rad18的连续遍在蛋白结合域识别多聚遍在蛋白的分子决定因素。

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摘要

Rad18 is a key factor in double-strand break DNA damage response (DDR) pathways via its association with K63-linked polyubiquitylated chromatin proteins through its bipartite ubiquitin-binding domains UBZ and LRM with extra residues between them. Rad18 binds K63-linked polyubiquitin chains as well as K48-linked ones and monoubiquitin. However, the detailed molecular basis of polyubiquitin recognition by UBZ and LRM remains unclear. Here, we examined the interaction of Rad18(201-240), including UBZ and LRM, with linear polyubiquitin chains that are structurally similar to the K63-linked ones. Rad18(201-240) binds linear polyubiquitin chains (Ub(2)Ub(4)) with affinity similar to that of a K63-linked one for diubiquitin. Ab initio modeling suggests that LRM and the extra residues at the C-terminus of UBZ (residues 227-237) likely form a continuous helix, termed the extended LR motif (ELRM). We obtained a molecular envelope for Rad18 UBZ-ELRM:linear Ub2 by small-angle X-ray scattering and derived a structural model for the complex. The Rad18:linear Ub(2) model indicates that ELRM enhances the binding of Rad18 with linear polyubiquitin by contacting the proximal ubiquitin moiety. Consistent with the structural analysis, mutational studies showed that residues in ELRM affect binding with linear Ub(2), not monoubiquitin. In cell data support the idea that ELRM is crucial in the localization of Rad18 to DNA damage sites. Specifically, E227 seems to be the most critical in polyubiquitin binding and localization to nuclear foci. Finally, we reveal that the ubiquitin-binding domains of Rad18 bind linear Ub(2) more tightly than those of RAP80, providing a quantitative basis for blockage of RAP80 at DSB sites. Taken together, our data demonstrate that Rad18(201-240) forms continuous ubiquitin-binding domains, comprising UBZ and ELRM, and provides a structural framework for polyubiquitin recognition by Rad18 in the DDR pathway at a molecular level.
机译：Rad18是双链断裂DNA损伤反应（DDR）途径中的关键因素，因为它通过二分体遍在蛋白结合域UBZ和LRM与K63连接的多泛素化染色质蛋白缔合，并且它们之间有额外的残基。 Rad18与K63连接的多泛素链以及K48连接的单泛素结合。但是，UBZ和LRM识别多聚泛素的详细分子基础仍然不清楚。在这里，我们检查了Rad18（201-240），包括UBZ和LRM，与线性聚泛素链的相互作用，该链的结构与K63连接的相似。 Rad18（201-240）结合线性多聚泛素链（Ub（2）Ub（4）），其亲和力类似于K63连接的双泛素。从头算建模表明，LRM和UBZ C末端的额外残基（残基227-237）可能形成连续的螺旋，称为扩展LR基序（ELRM）。我们通过小角X射线散射获得了Rad18 UBZ-ELRM：linear Ub2的分子包膜，并推导了该复合物的结构模型。 Rad18：linear Ub（2）模型表明ELRM通过接触近端泛素部分来增强Rad18与线性聚泛素的结合。与结构分析一致，突变研究表明ELRM中的残基影响与线性Ub（2）的结合，而不与单泛素结合。在细胞数据中，ELRM对于将Rad18定位于DNA损伤位点至关重要。具体而言，E227似乎是聚泛素结合和定位于核灶的最关键因素。最后，我们揭示了Rad18的泛素结合域比RAP80更紧密地结合线性Ub（2），为RAP80在DSB位点的阻断提供了定量基础。两者合计，我们的数据表明Rad18（201-240）形成连续的遍在蛋白结合域，包括UBZ和ELRM，并提供了Rad18在分子水平上通过Rad途径识别多遍在蛋白的结构框架。

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