通过基于 BRC 重复模块性的基序改组来改善 RAD51 结合物。
Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.
机构信息
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
Living Systems Institute, University of Exeter, Exeter EX4 4QD, United Kingdom.
出版信息
Proc Natl Acad Sci U S A. 2021 Nov 16;118(46). doi: 10.1073/pnas.2017708118.
Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.
蛋白质序列模块的交换支持进化过程中通过点突变无法获得的功能飞跃。在这里，我们研究了 BRCA2 中八个结合 BRC 重复序列内的两个 RAD51 相互作用模块的作用。我们通过打乱这些模块创建了 64 个嵌合重复序列，并测量了它们与 RAD51 的结合。我们发现某些打乱的模块组合比天然重复序列中的任何模块组合的结合能力都要强。令人惊讶的是，两个模块的贡献与天然重复序列的亲和力相关性较差，弱 BRC8 重复序列包含最有效的 N 端模块。与最强的天然重复序列 BRC4 相比，最强嵌合体 BRC8-2 与 RAD51 的结合提高了 -2.4 kCal/mol。RAD51:BRC8-2 复合物的晶体结构显示出该重复序列中界面适配的改善和β-发夹的扩展。BRC8-2 在人类细胞中具有功能，可防止电离辐射后核 RAD51 焦点的形成。
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