Incorporating intrinsically disordered regions into rationally designed proteins that target DNA

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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.