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Abstract
UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy. Recent studies indicate that interdomain interactions critically influence UHRF1's chromatin-binding properties, including allosteric regulation of its histone binding. Here, using an integrative approach that combines small angle X-ray scattering, NMR spectroscopy, and molecular dynamics simulations, we characterized the dynamics of the tandem tudor domain–plant homeodomain (TTD–PHD) histone reader module, including its 20-residue interdomain linker. We found that the apo TTD–PHD module in solution comprises a dynamic ensemble of conformers, approximately half of which are compact conformations, with the linker lying in the TTD peptide–binding groove. These compact conformations are amenable to cooperative, high-affinity histone binding. In the remaining conformations, the linker position was in flux, and the reader adopted both extended and compact states. Using a small-molecule fragment screening approach, we identified a compound, 4-benzylpiperidine-1-carboximidamide, that binds to the TTD groove, competes with linker binding, and promotes open TTD–PHD conformations that are less efficient at H3K9me3 binding. Our work reveals a mechanism by which the dynamic TTD–PHD module can be allosterically targeted with small molecules to modulate its histone reader function for therapeutic or experimental purposes.
Highlights
UHRF1 is a key mediator of inheritance of epigenetic DNA methylation patterns during cell division and is a putative target for cancer therapy
Hemimethylated DNA is recognized by the SRA domain (SET and RING-associated), whereas H3K9me3 marked chromatin is recognized by the plant homeodomain (PHD)3 and tandem tudor domain (TTD), which are connected by a 20-residue linker to form the tandem tudor domain–plant homeodomain (TTD–PHD) histone reader module
Large-scale intramolecular rearrangements play a critical role in UHRF1 function, consistent with a dynamic framework in which its conformational equilibria are shifted in response to the chromatin state and aggregate presence of other proteins and cellular factors with modulating influences (14 –16)
Summary
To assess the conformational heterogeneity of the TTD– PHD histone reader module, we used small angle X-ray scattering (SAXS) in solution. The Arg296-containing linker is bound within the TTD groove, and the mobility of the two domains is mediated largely by a 5-residue flexible “hinge” region (UHRF1297–301) (Fig. 1C). The first set (molecular dynamics pool-IN (MDPIN)) contains 6,000 TTD–PHD conformations generated with the linker bound to the TTD groove. The SAXS-fitted OEs largely reproduce the conformational space of their starting pools, but in comparison to each other, do not overlap, as would be expected if the TTD–PHD unit possessed restricted flexibility (supplemental Fig. S1). We used our two MD-generated pools of TTD–PHD structures (MDPIN and MDPIN/OUT) to carry out HYCUD-based predictions of the effective rotational correlation times of the TTD (TTD) and PHD (PHD). Using MDPIN, both PHD and TTD-predicted distributions are bellshaped curves with peaks positioned at ϳ 10 and 21 ns, respec-
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