Abstract
Methyl-CpG-binding proteins (MBPs) are selective readers of DNA methylation that play an essential role in mediating cellular transcription processes in both normal and diseased cells. This physiological function of MBPs has generated significant interest in understanding the mechanisms by which these proteins read and interpret DNA methylation signals. Zinc finger and BTB domain-containing 38 (ZBTB38) represents one member of the zinc finger (ZF) family of MBPs. We recently demonstrated that the C-terminal ZFs of ZBTB38 exhibit methyl-selective DNA binding within the ((A/G)TmCG(G/A)(mC/T)(G/A)) context both in vitro and within cells. Here we report the crystal structure of the first four C-terminal ZBTB38 ZFs (ZFs 6-9) in complex with the previously identified methylated consensus sequence at 1.75 Å resolution. From the structure, methyl-selective binding is preferentially localized at the 5' mCpG site of the bound DNA, which is facilitated through a series of base-specific interactions from residues within the α-helices of ZF7 and ZF8. ZF6 and ZF9 primarily stabilize ZF7 and ZF8 to facilitate the core base-specific interactions. Further structural and biochemical analyses, including solution NMR spectroscopy and electrophoretic mobility gel shift assays, revealed that the C-terminal ZFs of ZBTB38 utilize an alternative mode of mCpG recognition from the ZF MBPs structurally evaluated to date. Combined, these findings provide insight into the mechanism by which this ZF domain of ZBTB38 selectively recognizes methylated CpG sites and expands our understanding of how ZF-containing proteins can interpret this essential epigenetic mark.
Highlights
Methyl-CpG– binding proteins (MBPs) are selective readers of DNA methylation that play an essential role in mediating cellular transcription processes in both normal and diseased cells
Using a hybrid biophysical methodology and collective structural insight from available zinc finger (ZF)–methylated DNA structures, we previously presented an atomistic model in which it was proposed that a key arginine common to ZF MBPs was replaced with a lysine (Lys-1055) in methylated CpG (mCpG) recognition [37]
Initial attempts to crystallize the C-terminal ZFs of Zinc finger and BTB domain– containing 38 (ZBTB38) with the methylated C-terminal ZBTB38 binding sequence (mCZ38BS) focused on utilizing residues 1006 –1153, encompassing ZFs 6 –10 (Fig. 1A)
Summary
Optimization of the C-terminal ZBTB ZF domain for structural investigations with methylated DNA. Consistent with the solution NMR observations, ZFs 6 –10 and 6 –9 had nearly identical low nanomolar binding affinities (Kd values of ϳ5 nM) for mCZ38BS recognition, whereas ZFs 7–10 exhibited ϳ8-fold weaker binding (Fig. 1D and Fig. S1A) This finding was in agreement with our previously proposed atomistic model, where the ␣-helices of ZFs 6 –9 could be readily positioned along the major groove of the DNA, whereas the exact positioning of ZF10 remained indeterminant [37]. Overall, it appears that ZFs 6 and 9 primarily function to stabilize ZF7 and ZF8 for making core base–specific interactions
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