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, 289 (3), 1294-302

Probing the Dynamic Distribution of Bound States for Methylcytosine-Binding Domains on DNA

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Probing the Dynamic Distribution of Bound States for Methylcytosine-Binding Domains on DNA

Jason M Cramer et al. J Biol Chem.

Abstract

Although highly homologous to other methylcytosine-binding domain (MBD) proteins, MBD3 does not selectively bind methylated DNA, and thus the functional role of MBD3 remains in question. To explore the structural basis of its binding properties and potential function, we characterized the solution structure and binding distribution of the MBD3 MBD on hydroxymethylated, methylated, and unmethylated DNA. The overall fold of this domain is very similar to other MBDs, yet a key loop involved in DNA binding is more disordered than previously observed. Specific recognition of methylated DNA constrains the structure of this loop and results in large chemical shift changes in NMR spectra. Based on these spectral changes, we show that MBD3 preferentially localizes to methylated and, to a lesser degree, unmethylated cytosine-guanosine dinucleotides (CpGs), yet does not distinguish between hydroxymethylated and unmethylated sites. Measuring residual dipolar couplings for the different bound states clearly shows that the MBD3 structure does not change between methylation-specific and nonspecific binding modes. Furthermore, residual dipolar couplings measured for MBD3 bound to methylated DNA can be described by a linear combination of those for the methylation and nonspecific binding modes, confirming the preferential localization to methylated sites. The highly homologous MBD2 protein shows similar but much stronger localization to methylated as well as unmethylated CpGs. Together, these data establish the structural basis for the relative distribution of MBD2 and MBD3 on genomic DNA and their observed occupancy at active and inactive CpG-rich promoters.

Keywords: Biophysics; DNA Binding Protein; DNA Methylation; Hydroxymethylated DNA; MBD2; MBD3; Nuclear Magnetic Resonance; Protein Dynamics; Residual Dipolar Couplings.

Figures

FIGURE 1.
FIGURE 1.
Solution structure of MBD3 methyl-binding domain bound to hydroxymethylated DNA. A, stereo ribbon diagram (blue) of the MBD3 solution structure is shown for the ensemble of 20 calculated structures (Protein Data Bank code 2mb7). The loop connecting β2 and β3 (residues 24–33) is highlighted in light blue. B, ribbon diagram of the lowest energy solution structure is shown with key contact and chemical shift reporter residues depicted as sticks. C, per residue RMSD for backbone atoms is plotted for the solution structure ensemble of MBD3 (blue) and for the solution structure ensemble of cMBD2 (red) previously reported (Protein Data Bank code 2ky8) (18). D, the best fit protein alignment of the solution structures of cMBD2 (green) and MBD3 (blue) MBD is shown bound to the methylated DNA from the cMBD2-dsDNA solution structure (Protein Data Bank code 2ky8). E, diagram highlighting the cMBD2 hydrogen-bonding network while bound to methylated DNA and with key residues depicted as sticks. Structure diagrams were generated using the PyMOL program (Delano Scientific LLC).
FIGURE 2.
FIGURE 2.
Methyl-specific binding mode stabilizes a dynamic loop in MBD3. A, ribbon diagram of the MBD3 solution structure is shown and colored based on order parameters predicted from chemical shift index (S2; shading from blue to red reflects low to high). B, the predicted order parameters (S2) are plotted for the MBD3-hmCpG (black), MBD3-mCpG (blue dotted), and cMBD2-mCpG (red) complexes. C, bar plots are shown for the difference in order parameters (ΔS2) between the cMBD2-mCpG complex and MBD3-mCpG (black) and MBD3-hmCpG (gray) complexes. The loop connecting β2 and β3 (residues 24–33) is highlighted in light yellow in B and C.
FIGURE 3.
FIGURE 3.
Preferential localization of MBD3 to mCpG sites. A, bar plots show the chemical shift distances between MBD3-dsDNA complexes. B, an overlay of 15N HSQC spectra are shown for key reporter residues of MBD3 bound to CpG(×0), CpG(×1), CpG(×3), mCpG, and hmCpG as well as MBD3KY and cMBD2 bound to mCpG. C, the derivation of a simple statistical mechanical model for the distribution of MBD3 on mCpG (top panel) is shown with a mixed rendering diagram (bottom panel) depicting MBD3 docked onto a methylated site (red) as well as four nonmethylated sites (blue) of the mCpG DNA. Arrows indicate rapid exchange between these binding modes. D, overlays of 15N HSQC spectra are shown for key reporter residues of MBD3 and MBD3KY while bound to DNA of varying lengths.
FIGURE 4.
FIGURE 4.
Chemical shifts do not depend on concentration or the presence of hydroxymethylation. Overlays of 15N HSQC spectra are shown comparing MBD3-hmCpG (orange) (A) with MBD3-CpG(×3) and MBD3-mCpG at 600 μm (blue) and 300 μm (red) (B). Resonances for key reporter residues Ala30 and Gly27 are labeled.
FIGURE 5.
FIGURE 5.
MBD3 localizes to methylated DNA sites without significant conformational change. A and B, comparisons of measured 1DNH RDCs normalized to 2H2O quadrupole splitting of 10 Hz are plotted for MBD3-mCpG versus MBD3-CpG(×3) (A) and MBD3-mCpG versus MBD3KY-mCpG complexes (B). Red dotted ovals highlight those values that fall of the line of identity (gray line). C, the sum of squared residuals (SSR) is plotted as a function of ρm (Equation 3). The sum of squared residuals is minimized (red circle and arrow) at 37% mCpG bound (ρm = 0.37). D, plotting 1DNH RDCs for MBD3-mCpG observed versus predicted with ρm = 0.37 (Equation 3) shows good agreement with tight clustering around y = x. E, the measured 1DNH RDCs for each complex (MBD3-CpG(×3), MBD3KY-mCpG, and MBD3-mCpG, left to right plots, respectively) were fit to the solution structure of MBD3 by singular value decomposition, and the observed versus predicted values were plotted. The Q factors and correlation coefficients show good agreement with the solution structure indicating that the backbone structure of MBD3 does not change between complexes.
FIGURE 6.
FIGURE 6.
MBD2 distribution is influenced by DNA methylation status and CpG density. A, an overlay of 15N HSQC spectra for key reporter residues of MBD2 bound to CpG(×0), CpG(×1), CpG(×3) ± MBD3, mCpG, and hmCpG shows that cMBD2 preferentially localizes to DNA with mCpG, hmCpG, and multiple CpG sites and that localization is modified by the presence of equimolar MBD3. B, an overlay of 15N HSQC spectra cMBD2 bound to mCpG DNA of varying lengths confirms that cMBD2 strongly prefers mCpG sites.
FIGURE 7.
FIGURE 7.
MBD2 and MBD3 localization does not translate into a global binding affinity preference. Steady state surface plasmon resonance measurements are shown for MBD3 (top panel) and cMBD2 (bottom panel) binding to immobilized double-stranded oligonucleotides of varying CpG content and methylation status. The steady state response was normalized to the amount of DNA immobilized (Equation 1) such that the maximum response reflects the stoichiometry of binding.

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