Engineering an SspB-mediated degron for novel controllable protein degradation
Engineering an SspB-mediated degron for novel controllable protein degradation
复制标题
设计 SspB 介导的降解子以实现新型可控蛋白质降解
DOI：
10.1016/j.ymben.2022.10.013
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发表时间：
2022
影响因子：
8.4
通讯作者：
Tang Shuang-Yan
中科院分区：
文献类型：
--
作者：
Lei Yanyan;Chen Wei;Xiang La;Wu Jieyuan;Zhen Zhen;Jin Jian-Ming;Liang Chaoning;Tang Shuang-Yan
优雅的可控蛋白质降解工具在代谢工程和合成生物学设计中有着重要的应用。SspB介导的ClpXP蛋白水解系统被很好地表征，并且SspB充当将ssrA标记的底物拴系到ClpXP蛋白酶的接头。这种降解决定子被应用于代谢优化，但效率不令人满意。有限的高质量工具可用于可控的蛋白质降解。通过耦合结构导向建模和定向进化，我们建立了最先进的高通量筛选策略，用于工程化降解效率和这种降解决定子的SspB-ssrA结合特异性。我们的方法的可靠性证实了使用荧光测定和代谢工程的衣康酸或阿魏酸生物合成的SspB和ssrA突变体的功能验证。等温滴定量热分析和分子模拟表明，SspB和ssrA之间的适当而不是过于强烈的相互作用有利于降解效率。突变的SspB-ssrA对具有比野生型对高7-22倍的结合KD，导致更高的降解效率，揭示了定向进化在降解效率优化中优于理性设计。此外，还开发了与野生型SspB-ssrA对表现出低相互作用串扰的人工SspB-ssrA对。本研究的努力证明了SspB-ssrA结合口袋的可塑性，可用于设计高质量的可控蛋白质降解工具。获得的突变降解决定子丰富了代谢工程设计的工具箱。
Elegant controllable protein degradation tools have great applications in metabolic engineering and synthetic biology designs. SspB-mediated ClpXP proteolysis system is well characterized, and SspB acts as an adaptor tethering ssrA-tagged substrates to the ClpXP protease. This degron was applied in metabolism optimization, but the efficiency was barely satisfactory. Limited high-quality tools are available for controllable protein degradation. By coupling structure-guided modeling and directed evolution, we establish state-of-the-art high-throughput screening strategies for engineering both degradation efficiency and SspB-ssrA binding specificity of this degron. The reliability of our approach is confirmed by functional validation of both SspB and ssrA mutants using fluorescence assays and metabolic engineering of itaconic acid or ferulic acid biosynthesis. Isothermal titration calorimetry analysis and molecular modeling revealed that an appropriate instead of excessively strong interaction between SspB and ssrA benefited degradation efficiency. Mutated SspB-ssrA pairs with 7–22-fold higher bindingKDthan the wild-type pair led to higher degradation efficiency, revealing the advantage of directed evolution over rational design in degradation efficiency optimization. Furthermore, an artificial SspB-ssrA pair exhibiting low crosstalk of interactions with the wild-type SspB-ssrA pair was also developed. Efforts in this study have demonstrated the plasticity of SspB-ssrA binding pocket for designing high-quality controllable protein degradation tools. The obtained mutated degrons enriched the tool box of metabolic engineering designs.