Incorporating intrinsically disordered regions into rationally designed proteins that target DNA

自1995年以来，我们一直专注于蛋白质：DNA设计。我们从探索二聚体bZIP转录因子基序(碱性区域/亮氨酸拉链，60个氨基酸/单体)开始，然后转移到bHLH和bHLHZ基序(碱性区域/螺旋-环-螺旋/亮氨酸拉链，分别为68和92个氨基酸/单体)。这些DNA结合域普遍存在于转录因子中，大多是α-螺旋的，并以高亲和力和序列特异性与其DNA靶标结合。TFS通常包含固有的无序区(IDR)。这些IDR在晶体结构上是无序的，与原核生物相比，在人类和真核生物蛋白中更为丰富。有趣的是，PDB(蛋白质数据库)包含比真核/人类更多的原核结构。此外，与其他人类蛋白质相比，人Tf在PDB中的代表性较低，这表明获得高无序性蛋白质的高分辨率结构是一项挑战-人Tf中49%的残基位于IDR中。IDR不仅极大地改变了我们对蛋白质与目标相互作用的理解，而且挑战了我们使用蛋白质支架进行设计的能力。进化使蛋白质成为催化、识别、信号和许多其他功能的首选分子。因此，我们在自己的分子设计中使用蛋白质框架。我们依靠晶体结构来帮助我们进行合理的设计工作。最近，我们正在通过持续进化来采用“非理性”设计，因为这种方法可以解释无序的区域。事实证明，IDR是一种多功能结构，具有微调基因调控的微妙能力。因此，我们建议在我们的蛋白质设计程序中使用IDR，并使用PACE(噬菌体辅助的持续进化)来设计IDR。作为一名研究生，我研究过含有对DNA结合功能至关重要的无序区域的DNA结合区域。当时，我们不明白无序区域如何发挥如此关键的作用，因为晶体结构显示出折叠良好的螺旋基序结合了DNA主槽。我们现在知道这些重要的地区是IDR。有了几十年的新知识，我可以重新审视这个问题--这是一个经典的老生常谈的例子。研究生Serban Popa和Duan Tan将引领我们的目标，并指导两名本科生。我的学员在细胞生物学(酵母、细菌)、生物物理学(定量测量、光谱分析)、分析化学(分离)、定向进化和噬菌体工作(2018年诺贝尔奖)方面获得了真正的多学科培训，这些都对解决21世纪的研究问题至关重要。多年来，我们已经成功地使用Rational Design设计出了与天然Tf功能相媲美的蛋白质。将我们成熟的理性设计能力与新开发的非理性定向进化相结合，将使我们设计具有所需结构和功能的最低限度蛋白质的长期目标成为可能。

We have focused on protein:DNA design since 1995. We started by exploring the dimeric bZIP transcription factor motif (basic region/leucine zipper, 60 amino acids/monomer), and then moved to the bHLH and bHLHZ motifs (basic region/helix-loop-helix/leucine zipper, 68 and 92 amino acids/monomer, respectively). These DNA-binding domains are commonly found in transcription factors (TFs), are mostly alpha-helical, and bind to their DNA targets with high affinity and sequence specificity. TFs often contain intrinsically disordered regions (IDRs). These IDRs are disordered in crystal structures and more abundant in human and eukyarotic proteins compared to prokaryotic. Interestingly, the PDB (Protein Data Bank) contains more prokaryotic structures than eukaryotic/human. Moreover, human TFs have low representation in the PDB compared with other human proteins, indicating the challenge of gaining high-resolution structures of proteins with high disorder-49% of residues in human TFs are in IDRs. IDRs not only dramatically change our understanding of proteins interacting with targets, but also they challenge our ability to use the protein scaffold for design. Evolution produced proteins as the molecule-of-choice for catalysis, recognition, signalling, and many other functions. Thus, we use the protein framework in our own molecular design. We depend on crystal structures to aid our rational design efforts. More recently, we are adopting "nonrational" design via continuous evolution, as such methods can account for disordered regions. IDRs are proving to be versatile structures with exquisite ability to fine-tune gene regulation. Thus, we propose to use IDRs in our protein design program and use PACE (phage assisted continuous evolution) for IDR design. As a grad student, I studied DNA-binding domains harbouring disordered regions that were vital to DNA-binding function. At the time, we did not understand how a disordered region could play such a critical role, given that crystal structures showed well-folded helical motifs binding the DNA major groove. We now know these important regions are IDRs. With decades of new knowledge, I can revisit this problem-this is a classic example of what-is-old-is-new-again. Grad students Serban Popa and Duan Tan will spearhead our objectives and guide two undergrads. My trainees gain a truly multidisciplinary training in cell biology (yeast, bacteria), biophysics (quantitative measurements, spectrometry), analytical chemistry (separations), directed evolution and phage work (Nobel Prize 2018), which are all critical toward solving the research problems of the 21st century. We have successfully used rational design for many years to design proteins that rival native TF function. Combining our mature rational design capability with newly developing nonrational directed evolution will make possible our longer-term goal of design of minimalist proteins with desired structure and function.