Exploiting the recognition code for elucidating the mechanism of zinc finger protein-DNA interactions

Engineering zinc finger protein motifs for specific binding to double-stranded DNA is critical for targeted genome editing. Most existing tools for predicting DNA-binding specificity in zinc fingers are trained on data obtained from naturally occurring proteins, thereby skewing the predictions. Moreover, these mostly neglect the cooperativity exhibited by zinc fingers.

Here, we present an ab-initio method that is based on mutation of the key α-helical residues of individual fingers of the parent template for Zif-268 and its consensus sequence (PDB ID: 1AAY). In an attempt to elucidate the mechanism of zinc finger protein-DNA interactions, we evaluated and compared three approaches, differing in the amino acid mutations introduced in the Zif-268 parent template, and the mode of binding they try to mimic, i.e., modular and synergistic mode of binding.

Comparative evaluation of the three strategies reveals that the synergistic mode of binding appears to mimic the ideal mechanism of DNA-zinc finger protein binding. Analysis of the predictions made by all three strategies indicate strong dependence of zinc finger binding specificity on the amino acid propensity and the position of a 3-bp DNA sub-site in the target DNA sequence. Moreover, the binding affinity of the individual zinc fingers was found to increase in the order Finger 1 < Finger 2 < Finger 3, thus confirming the cooperative effect.

Our analysis offers novel insights into the prediction of ZFPs for target DNA sequences and the approaches have been made available as an easy to use web server at http://web.iitd.ac.in/~sundar/zifpredict_ihbe.