The domain architecture of the protozoan protein J-DNA–binding protein 1 suggests synergy between base J DNA binding and thymidine hydroxylase activity

Athanassios Adamopoulos,Tatjana Heidebrecht,Jeroen Roosendaal,Wouter G Touw,Isabelle Q Phan,Jos Beijnen,Anastassis Perrakis

doi:10.1074/jbc.ra119.007393

Abstract

J-DNA-binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (β-d-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosoma and Leishmania JBP1 has two known functional domains: an N-terminal T 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 structure. We found that this Δ-JDBD domain retains TH activity in vitro but displays a 15-fold lower apparent rate of hydroxylation compared with JBP1. Small-angle X-ray scattering (SAXS) experiments on JBP1 and JDBD in the presence or absence of J-DNA and on Δ-JDBD enabled 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 T hydroxylation 12-14 bp downstream on the complementary strand of the J-recognition site. We postulate that insertion of the JDBD module into the Δ-JDBD scaffold during evolution provided a mechanism that synergized J recognition and T hydroxylation, ensuring inheritance of base J in specific sequence patterns following DNA replication in kinetoplastid parasites.

Highlights

J-DNA– binding protein 1 (JBP1) contributes to the biosynthesis and maintenance of base J (␤-D-glucosyl-hydroxymethyluracil), an epigenetic modification of thymidine (T) confined to pathogenic protozoa such as Trypanosoma and Leishmania
As J-DNA– binding domain (JDBD) is in the middle of the JBP1 sequence (Fig. 1B), we decided to test the possibility that the JDBD is an insertion domain into a “T hydroxylase (TH) domain” fold that spans the rest of the JBP1 sequence
When we run the cleaved protein on an size-exclusion chromatography (SEC) column in the absence of detergent, the elution profile showed a single symmetric peak (Fig. 1D) of approximately the same molecular weight as ⌬-JDBD, whereas the SDS-PAGE analysis of the eluted fractions confirmed that this peak has both bands present

Summary

Results

Countless previous attempts to truncate JBP1 either C-terminally or N-terminally (before or after the JDBD), to obtain the N-terminal TH domain or a putative C-terminal domain, had invariably failed to yield soluble protein in our hands. The observed diffusion in scattering contrast and the reduced packing density in the WT, full-length JBP1 compared with ⌬-JBP1 suggest that the JDBD domain is flexible with respect to the ⌬-JDBD scaffold To validate this hypothesis, we decided to create ab initio three-dimensional models based on the SAXS data. If ⌬-JDBD is viewed as an ellipsoid, the JDBD is consistently positioned toward one half of the ellipsoid but adopts multiple conformations around the long axis of the ellipsoid This analysis is compatible with the model-independent analysis of the SAXS data and strengthens our previous hypothesis that the JDBD domain is flexible with respect to the ⌬-JDBD scaffold. The complex between J-23-DNA and JBP1 eluted from the SAXS column as a single peak and was confirmed by the Details for the phases used in MONSA for modeling and all resulting clusters C-1–C-5) for all complexes and subcomplexes used in this study

C-1 C-2 C-3 C-4 C-5

Discussion

Experimental procedures