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。我们首先研究了二聚体bZIP转录因子基序（基本区/亮氨酸拉链，60个氨基酸/单体），然后研究了bHLH和bHLHZ基序（基本区/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结合结构域常见于转录因子（tf）中，大多呈α -螺旋状，并以高亲和力和序列特异性结合其DNA靶标。tf通常包含内在无序区（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，人类tf在PDB中的代表性较低，这表明获得高无序度蛋白质的高分辨率结构的挑战-人类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靶标相互作用的理解，而且还挑战了我们使用蛋白质支架进行设计的能力。进化产生的蛋白质作为催化、识别、信号传导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了蛋白质框架。我们依靠晶体结构来帮助我们进行合理的设计。最近，我们正在通过持续进化采用“非理性”设计，因为这样的方法可以解释无序区域。idr被证明是一种多功能结构，具有微调基因调控的精细能力。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（噬菌体辅助连续进化）进行IDR设计。作为一名研究生，我研究了dna结合域，其中包含对dna结合功能至关重要的无序区域。当时，我们不明白一个无序区域如何发挥如此关键的作用，因为晶体结构显示出与DNA主槽结合的折叠良好的螺旋基序。我们现在知道这些重要的区域是idr。有了几十年的新知识，我可以重新审视这个问题——这是“旧即新”的经典例子。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、光谱法）、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，这些都是解决21世纪研究问题的关键。多年来，我们已经成功地使用理性设计来设计与天然TF功能相媲美的蛋白质。将我们成熟的理性设计能力与新发展的非理性定向进化相结合，将使我们设计具有理想结构和功能的极简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.