JBP1 (J-DNA Binding Protein 1) contributes to biosynthesis and maintenance of base J (β-D-glucosyl-hydroxymethyluracil), a modification of thymidine (T) confined to pathogenic protozoa. JBP1 has two known functional domains: an N-terminal thymidine hydroxylase (TH) homologous to the 5-methylcytosine hydroxylase domain in TET proteins; and a J-DNA binding domain (JDBD) that resides in the middle of JBP1. Here we show that removing JDBD from JBP1 results in a soluble protein (Δ-JDBD) with the N- and C-terminal regions tightly associated together in a well-ordered domain. This Δ-JDBD domain retains thymidine hydroxylation activity in vitro, but displays a fifteen-fold lower apparent rate of hydroxylation compared to JBP1. Small Angle X-ray Scattering (SAXS) experiments on JBP1 and JDBD in the presence and absence of J-DNA, and on Δ-JDBD, allowed us to generate low-resolution three-dimensional models. We conclude that Δ-JDBD, and not the N-terminal region of JBP1 alone, is a distinct folding unit. Our SAXS-based model supports the notion that binding of JDBD specifically to J-DNA can facilitate hydroxylation a T 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module in the Δ-JDBD scaffold during evolution provided a mechanism to synergize between J recognition and T hydroxylation, ensuring inheritance of J in specific sequence patterns following DNA replication.
