JBP1 and JBP3 have conserved structures but different affinity to base-J

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Base-J (β-D-glucopyranosyloxymethyluracil) is a unique DNA modification found in kinetoplastids.
• This modification is recognized by specific DNA-binding proteins, JBP1 and JBP3.
• Both JBP1 and JBP3 utilize a conserved J-DNA binding domain (JDBD) for recognition.

Purpose of the Study:

• To investigate the differential binding affinities of JDBD-JBP1 and JDBD-JBP3 to J-DNA.
• To elucidate the structural basis for the distinct DNA recognition properties of JBP1 and JBP3.
• To identify key structural elements involved in J-DNA binding by JBPs.

Main Methods:

• Comparative analysis of J-DNA binding affinities using JDBD-JBP1 and JDBD-JBP3.
• Determination and comparison of crystal structures of JDBD-JBP3 and JDBD-JBP1.
• Site-directed mutagenesis to assess the role of specific amino acid residues in DNA binding.

Main Results:

• JDBD-JBP3 exhibits significantly lower affinity (approx. 1,000-fold weaker) for J-DNA compared to JDBD-JBP1.
• JDBD-JBP1 discriminates between J-DNA and unmodified DNA with a factor of ~10,000, while JDBD-JBP3 discriminates with a factor of ~5.
• Structural comparison revealed a flexible α5-helix in JDBD-JBP3 lacking a positively charged patch present in JDBD-JBP1, which is crucial for DNA binding.

Conclusions:

• The positively charged patch on the α5-helix of JDBD-JBP1 is essential for high-affinity J-DNA binding.
• The α5-helix likely undergoes conformational changes upon JBP1 binding to stabilize the complex.
• Understanding JBP-J-DNA interactions provides insights into kinetoplastid biology and potential therapeutic targets.