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

Levani Zandarashvili, Alexandre Esadze, Dana Vuzman, Catherine A. Kemme, Yaakov Levy, Junji Iwahara

    Research output: Contribution to journalArticle

    49 Citations (Scopus)

    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.

    Original languageEnglish (US)
    Pages (from-to)E5142-E5149
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume112
    Issue number37
    DOIs
    StatePublished - Sep 15 2015

    Fingerprint

    Zinc Fingers
    DNA
    DNA-Binding Proteins
    Proteins
    Synthetic Genes
    DNA Cleavage
    Thermodynamics
    Mutagenesis
    Transcription Factors
    Magnetic Resonance Spectroscopy
    Fluorescence
    Technology
    Enzymes
    Gene Editing

    Keywords

    • DNA scanning
    • Dynamics
    • Kinetics
    • Protein-DNA interactions
    • Target search

    ASJC Scopus subject areas

    • General

    Cite this

    Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble. / Zandarashvili, Levani; Esadze, Alexandre; Vuzman, Dana; Kemme, Catherine A.; Levy, Yaakov; Iwahara, Junji.

    In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 112, No. 37, 15.09.2015, p. E5142-E5149.

    Research output: Contribution to journalArticle

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