Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

Abstract

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 DNAscanning 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.protein-DNA interactions | DNA scanning | target search | kinetics | dynamics A rtificial transcription factors and DNA-modifying enzymes have gained popularity as powerful means for artificial gene regulation and genome editing (1-7). Successful applications were reported on artificial zinc-finger (ZF) proteins engineered to exhibit a desired sequence specificity in binding to DNA (1-5). Artificial ZF transcription factors, comprising engineered ZFs and transactivation or repression domains, are used to regulate particular genes (1-3). ZF nucleases (ZFNs), comprising engineered ZFs and a FokI nuclease domain (ND), can site-specifically cleave DNA at particular sequences and allow for genome editing in vivo (4, 5). ZFN-based gene therapy for HIV infection is currently under phase 2 clinical trials, yielding some successful cases (8). Other studies suggested that ZF-based gene control could also be therapeutically effective for hemophilia (9) and Parkinson's disease (10). For the success of these technologies, however, two issues, toxicity and low efficiency, should be resolved (4, 5, 11). Regarding the latter issue, some studies (11)(12)(13)(14) suggest that, despite high affinities for target DNA, the artificial proteins do not necessa...

Discrete-State Kinetics Model for NMR-Based Analysis of Protein Translocation on DNA at Equilibrium

“…This estimate is consistent with typical D 1 values determined by fluorescence methods for other relatively small proteins. 9,20,33 …”

“…Previous biophysical or biochemical studies showed that D 1 is typically on the order of 10 4 –10 6 bp 2 s −1 (e.g., see Refs. 9,20,26–29 ).…”

Structural Insights into c-Myc-interacting Zinc Finger Protein-1 (Miz-1) Delineate Domains Required for DNA Scanning and Sequence-specific Binding

“…8B, the Miz1-4 construct binds to all of the sequences tested, including two unrelated and nonspecific sequences, with a similar low apparent affinity in the micromolar range. Reported affinities for series of three ZFs to nonspecific DNA sequences normally lie in the low micromolar range, whereas their specific binding rather lies in the nanomolar to picomolar range (17,(32)(33)(34). Hence, our data clearly demonstrate that Miz-1 ZFs 1-4 bind DNA with an affinity and a specificity that are not sufficient to promote specific molecular recognition of the consensus DNA or the p15 core promoter regions by this transcription factor.…”

“…However, because those recognition modes involve ZFs 1-6 and the matches do not correlate well with the conserved regions, we believe that these binding modes, although less stable, could serve in the DNA scanning. In this regard, Zandarashvili et al (34,36) recently published studies demonstrating the importance of the presence of a lower affinity DNA binding ZF for optimal target search efficiency by a protein containing three ZFs. Analogously, we propose that ZFs 1-6 serve a similar purpose for the efficient recognition of the consensus by subsets of ZFs 7-12.…”

Optimal Length of Conformational Transition Region in Protein Search for Targets on DNA

“…Experiments also indicate that the nonspecifically bound proteins are involved in conformational transitions between searching conformations with weak protein–DNA interactions, when the proteins slide quite fast along DNA, and stronger interacting recognition conformations, when the proteins move much slower. 17,20,21,23,31 The protein molecule can identify the specific target site only in the recognition mode. However, it seems that these conformational fluctuations should make the search less efficient.…”