Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble.
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.
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch at Galveston, Galveston, TX 77555;
Levani
L
Zandarashvili
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch at Galveston, Galveston, TX 77555;
Alexandre
A
Esadze
Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel.
Dana
D
Vuzman
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch at Galveston, Galveston, TX 77555;
Catherine A
CA
Kemme
Department of Structural Biology, Weizmann Institute of Science, Rehovot, 76100, Israel.
Yaakov
Y
Levy
Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch at Galveston, Galveston, TX 77555; j.iwahara@utmb.edu.
Junji
J
Iwahara
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry*; D051766:Early Growth Response Protein 1 / Q000737:chemistry*; D016678:Genome; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D055672:Static Electricity; D013816:Thermodynamics; D016335:Zinc Fingers
C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; D004247:DNA
DNA scanning; dynamics; kinetics; protein–DNA interactions; target search