期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：37
页码：E5142-E5149
DOI：10.1073/pnas.1507726112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMany transcription factors and DNA repair/modifying enzymes must first locate the target sites through stochastic scanning of DNA in the vast presence of nonspecific sites. In this work, we demonstrate that target search by these proteins can be accelerated via engineering based on structural dynamic knowledge of the DNA-scanning process. Our biophysical data for the Egr-1 zinc-finger protein and its nuclease derivatives reveal kinetic and thermodynamic roles of the conformational equilibrium between two modes in the DNA-scanning process: one suitable for search and the other for recognition. We found that optimizing the balance between the search and recognition modes improves efficiency in zinc-finger proteins' target search. This finding can help advance zinc-finger technology for artificial gene regulation and genome editing. Although engineering of transcription factors and DNA-modifying enzymes has drawn substantial attention for artificial gene regulation and genome editing, most efforts focus on affinity and specificity of the DNA-binding proteins, typically overlooking the kinetic properties of these proteins. However, a simplistic pursuit of high affinity can lead to kinetically deficient proteins that spend too much time at nonspecific sites before reaching their targets on DNA. We demonstrate that structural dynamic knowledge of the DNA-scanning process allows for kinetically and thermodynamically balanced engineering of DNA-binding proteins. Our current study of the zinc-finger protein Egr-1 (also known as Zif268) and its nuclease derivatives reveals kinetic and thermodynamic roles of the dynamic conformational equilibrium between two modes during the DNA-scanning process: one mode suitable for search and the other for recognition. By mutagenesis, we were able to shift this equilibrium, as confirmed by NMR spectroscopy. Using fluorescence and biochemical assays as well as computational simulations, we analyzed how the shifts of the conformational equilibrium influence binding affinity, target search kinetics, and efficiency in displacing other proteins from the target sites. A shift toward the recognition mode caused an increase in affinity for DNA and a decrease in search efficiency. In contrast, a shift toward the search mode caused a decrease in affinity and an increase in search efficiency. This accelerated site-specific DNA cleavage by the zinc-finger nuclease, without enhancing off-target cleavage. Our study shows that appropriate modulation of the dynamic conformational ensemble can greatly improve zinc-finger technology, which has used Egr-1 (Zif268) as a major scaffold for engineering.
