Characterization of a De Novo DNA-Binding Protein Targeting Five Operator Sites in the 933W Phage

Abstract

We characterized a series of de novo proteins that each bind to five unique DNA sequences, offering insight into the minimal engineering requirements for this function. This was investigated via the 933W lambdoid phage associated with food poisoning from shiga toxin producing E. coli. When 933W infects E. coli, there are two possible life cycles: lytic and lysogenic. The genetic switch between these two pathways is controlled by a DNA-binding protein that recognizes five unique sequences. Activation of the lytic pathway via the Cro protein results in toxin production and host cell death. Activation of the lysogenic pathway via the CI protein integrates the genome into the host’s genome, where the virus is dormant and no toxin is produced. This project characterizes our efforts to engineer a de novo DNA-binding protein that outcompetes the native CI and Cro proteins at all five DNA sites. The proteins were designed with a recognition helix from the 933W CI protein, incorporated diagonally into a doubled four-helix bundle. This was named Double Positive Mutant Diagonal (DPMD). DPMD was expressed via recombinant DNA techniques, and its structure was verified by circular dichroism (CD). Surface Plasmon Resonance (SPR) was used to assess DPMD’s binding affinities to three sequences: 933WOR1, 933WOR2, and 933WOR3. DPMD binds to 933WOR1 with a KD of 1.76 nM, to 933WOR2 with a KD of 3.16 nM. Current work focuses on collecting SPR data for 933WOR3. Future experiments will compare variants with altered recognition helix placement.