Structural basis of tandemly connected engrailed homeodomains for designing an array of DNA-binding proteins

Widely used genome-editing enzymes such as TALEN and CRISPR have a high molecular weight, which makes them difficult to be delivered to cells. To develop a novel genome-editing enzyme with a smaller molecular weight, we have focused on engrailed homeodomain protein (EHD). In our previous work, we have successfully created a novel DNA-binding protein by connecting two EHD domains with a linker (EHD2). The protein specifically recognizes the tandemly connected target site of the individual EHD’s target site in E.coli cells. The specific recognition for the tandem target sequence was achieved only when the conserved arginine 53 of the EHD was mutated to alanine in the tandem proteins ((EHD[R53A])2). The tandem proteins without that mutation mostly bound to sequences containing the target of the monomeric EHD. To reveal the molecular mechanisms for recognition of the tandem target site, we determined a crystal structure of ((EHD[R53A])2-DNA complex. We found that base-specific interactions of I47, K50 and N51 with DNA in the major groove observed in the wt were completely preserved in ((EHD[R53A])2. Together with the biological functional analysis, we conclude that ((EHD[R53A])2 realizes the precise recognition of the tandem target sites in cell by concurrently two individual EHD domains’ binding to the target sites.3rd QST International Symposiu