Clark-ElSayed Alia, Creed Ethan, Nayvelt Kaila, Ellington Andrew
Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA.
bioRxiv. 2025 Jul 11:2025.07.10.663684. doi: 10.1101/2025.07.10.663684.
Recently, the number of ML-based tools for protein design has greatly expanded. Although there have been many successful uses of these tools for improved stability, solubility, and ligand binding, there have been fewer uses of these tools for designing proteins that have intrinsic allosteric mechanisms. In this regard, allosteric transcription factors (aTFs) are a class of regulatory proteins that includes repressors and activators that respond to environmental signals by allosteric communication to regulate their binding with DNA elements. The data exist for evaluating design algorithms for their ability to take allostery into account, as many aTFs have previously been engineered to respond to new ligands, enabling their use as biosensors. In particular, previous work from our lab used directed evolution to change the effector specificity of the transcriptional repressor, RamR, from cholic acids to each of five benzylisoquinoline alkaloids (BIAs). We wanted to see to what extent we could recapitulate these results by instead using LigandMPNN to design the ligand binding pocket. The wild-type RamR structure was predicted in complex with the five BIAs, and the binding pocket was then targeted for computational redesign. However, there was little overlap between the results of directed evolution and computational redesign, and in fact the nine redesigned protein variants tested proved not to be functional in . Overall, these and other results suggest that different protein design methods may be needed to advance the computational design of allosteric or conformationally flexible proteins.
最近，基于机器学习的蛋白质设计工具数量大幅增加。尽管这些工具在提高稳定性、溶解性和配体结合方面有许多成功应用，但在设计具有内在变构机制的蛋白质方面应用较少。在这方面，变构转录因子（aTFs）是一类调节蛋白，包括阻遏物和激活物，它们通过变构通讯响应环境信号来调节与DNA元件的结合。由于许多aTFs此前已被改造以响应新配体，从而能够用作生物传感器，因此存在评估设计算法考虑变构能力的数据。特别是，我们实验室之前的工作利用定向进化将转录阻遏物RamR的效应物特异性从胆酸改变为五种苄基异喹啉生物碱（BIAs）中的每一种。我们想看看通过使用LigandMPNN设计配体结合口袋，能在多大程度上重现这些结果。预测了野生型RamR与五种BIAs结合的结构，然后针对结合口袋进行计算重新设计。然而，定向进化和计算重新设计的结果几乎没有重叠，事实上，测试的九个重新设计的蛋白质变体在……中被证明没有功能。总体而言，这些结果和其他结果表明，可能需要不同的蛋白质设计方法来推进变构或构象灵活蛋白质的计算设计。
