Solution structure and dynamic analysis of chicken MBD2 methyl binding domain bound to a target-methylated DNA sequence

Abstract

The epigenetic code of DNA methylation is interpreted chiefly by methyl cytosine binding domain (MBD) proteins which in turn recruit multiprotein co-repressor complexes. We previously isolated one such complex, MBD2-NuRD, from primary erythroid cells and have shown it contributes to embryonic/fetal β-type globin gene silencing during development. This complex has been implicated in silencing tumor suppressor genes in a variety of human tumor cell types. Here we present structural details of chicken MBD2 bound to a methylated DNA sequence from the ρ-globin promoter to which it binds in vivo and mediates developmental transcriptional silencing in normal erythroid cells. While previous studies have failed to show sequence specificity for MBD2 outside of the symmetric mCpG, we find that this domain binds in a single orientation on the ρ-globin target DNA sequence. Further, we show that the orientation and affinity depends on guanine immediately following the mCpG dinucleotide. Dynamic analyses show that DNA binding stabilizes the central β-sheet, while the N- and C-terminal regions of the protein maintain mobility. Taken together, these data lead to a model in which DNA binding stabilizes the MBD2 structure and that binding orientation and affinity is influenced by the DNA sequence surrounding the central mCpG.

Figure 1.

Solution structure of cMBD2 methyl binding domain bound to methylated DNA. (a) A best-fit superimposition licorice diagram of protein backbone (cyan) and DNA heavy atoms (blue) is shown for the ensemble of 20 calculated structures. (b) A stereo cartoon diagram of a representative MBD2 (cyan) and DNA (blue/orange) is shown. (c) A detailed line diagram of the protein:DNA interface is depicted with contacting protein residues (cyan) and mCpG DNA bases (yellow) shown as sticks. (d) A diagram depicting base-specific (solid lines) and phosphate backbone (dashed lines) contacts between MBD2 and DNA (for simplicity, only the central 6 bp are shown). Structure figures were generated with (a) VMD-XPLOR (65) and (b, c) PyMOL (Delano Scientific, LLC).

Figure 2.

Orientation preference for MBD2 bound to methylated ρ-globin DNA sequence. (a) 3D ¹⁵N-HSQC-NOESY slices corresponding to the N_ε-H_ε of Arg24 and Arg46 of MBD2 when bound to wild-type and inverted DNA sequences. NOE crosspeaks for mCyt105H5 and mCyt115H5 are labeled. (b) PRE Q values are calculated over an ensemble of MBD2 orientations for data obtained when bound to wild-type (solid line) and inverted (dashed line) DNA sequences. The ensemble consists of 10 copies of MBD2 with 0–10 of these having a reversed orientation with respect to the DNA. (c) A cartoon diagram shows the two alternative MBD2 orientations that make up the ensemble. The orientation as solved by NMR for wild-type DNA (cyan) and a symmetrically reversed orientation (yellow) are depicted.

Figure 3.

Binding affinity of cMBD2 to methylated DNA. (a) Surface plasmon resonance analysis for varying concentrations of wild-type cMBD2 binding to a 3′-biotinylated and methylated 10 bp target sequence coupled to a Sensor Chip SA on a Biacore T100 (GE Healthcare). Steady state binding response was analyzed for varying concentrations of cMBD2 with (b) specific point mutations or (c) binding to modified DNA sequences. The data were fit using the Biacore T100 evaluation software. For comparison, the response units for each were normalized to an R_max = 100 (Table 2).

Figure 4.

¹⁵N-relaxation dynamic analysis of cMBD2 bound to DNA. (a) Model free parameters (S², τ_e and R_ex) derived from ¹⁵N-relaxation data are plotted against residue number. (b) A stereo cartoon diagram of the cMBD2-DNA structure is shown and colored according to generalized order parameter from high (blue) to low (red), with proline residues (not included in the analysis) colored dark gray and residues with broadened amide resonances (not included, but likely to undergo slow exchange motions) colored peach. Structurally equivalent residues for some of the most common Rett syndrome missense mutations are depicted as spheres.