Department of Biological Sciences, The University of Texas at Dallas, Dallas, TX, USA.
Department of Bioinformatics and Computational Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
Nat Commun. 2021 Sep 22;12(1):5592. doi: 10.1038/s41467-021-25851-6.
Genetic sensors with unique combinations of DNA recognition and allosteric response can be created by hybridizing DNA-binding modules (DBMs) and ligand-binding modules (LBMs) from distinct transcriptional repressors. This module swapping approach is limited by incompatibility between DBMs and LBMs from different proteins, due to the loss of critical module-module interactions after hybridization. We determine a design strategy for restoring key interactions between DBMs and LBMs by using a computational model informed by coevolutionary traits in the LacI family. This model predicts the influence of proposed mutations on protein structure and function, quantifying the feasibility of each mutation for rescuing hybrid repressors. We accurately predict which hybrid repressors can be rescued by mutating residues to reinstall relevant module-module interactions. Experimental results confirm that dynamic ranges of gene expression induction were improved significantly in these mutants. This approach enhances the molecular and mechanistic understanding of LacI family proteins, and advances the ability to design modular genetic parts.
通过杂交来自不同转录阻遏物的 DNA 结合模块 (DBM) 和配体结合模块 (LBM)，可以创建具有独特 DNA 识别和变构反应组合的遗传传感器。由于杂交后关键的模块-模块相互作用丢失，这种模块交换方法受到来自不同蛋白质的 DBM 和 LBM 之间不兼容的限制。我们通过使用受 LacI 家族共进化特征启发的计算模型来确定一种设计策略，用于通过使用受 LacI 家族共进化特征启发的计算模型来恢复 DBM 和 LBM 之间的关键相互作用。该模型预测了提议突变对蛋白质结构和功能的影响，量化了每种突变恢复杂交阻遏物的可行性。我们准确地预测了通过突变残基重新安装相关模块-模块相互作用可以恢复哪些杂交阻遏物。实验结果证实，这些突变体中基因表达诱导的动态范围显著提高。这种方法增强了对 LacI 家族蛋白的分子和机械理解，并提高了设计模块化遗传元件的能力。
