Structural basis for designing an array of engrailed homeodomains

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Small DNA-binding proteins are crucial scaffolds for molecular tools like genome editing.
• Engrailed homeodomain (EHD) is a small DNA-binding protein with potential for engineering.

Purpose of the Study:

• To investigate the mechanism of tandem engrailed homeodomains (EHD[R53A])2 in recognizing longer DNA target sequences.
• To evaluate the potential of engineered EHDs as molecular modules for DNA targeting.

Main Methods:

• Determined the crystal structure of the (EHD[R53A])2-DNA complex at 1.6 Å resolution.
• Performed bacterial assays to confirm DNA-binding interactions under cellular conditions.

Main Results:

• The crystal structure revealed that individual EHDs maintain their typical homeodomain fold.
• Base-specific interactions in the major groove, critical for wild-type EHD affinity/specificity, are preserved in (EHD[R53A])2.
• The R53A mutation reduces DNA-binding affinity by disrupting arginine-phosphate interactions but allows precise recognition of tandem target sites.

Conclusions:

• Engineered tandem EHDs, specifically (EHD[R53A])2, precisely recognize tandem DNA target sites within cells.
• Modulating the binding activity of individual EHDs is essential for constructing protein arrays for multi-site DNA recognition.
• This work provides insights into designing protein arrays for targeted DNA manipulation.