Analysis of the complex between MBD2 and the histone deacetylase core of NuRD reveals key interactions critical for gene silencing

Abstract

The nucleosome remodeling and deacetylase (NuRD) complex modifies nucleosome positioning and chromatin compaction to regulate gene expression. The methyl-CpG-binding domain proteins 2 and 3 (MBD2 and MBD3) play a critical role in complex formation; however, the molecular details of how they interact with other NuRD components have yet to be fully elucidated. We previously showed that an intrinsically disordered region (IDR) of MBD2 is necessary and sufficient to bind to the histone deacetylase core of NuRD. Building on that work, we have measured the inherent structural propensity of the MBD2-IDR using solvent and site-specific paramagnetic relaxation enhancement measurements. We then used the AlphaFold2 machine learning software to generate a model of the complex between MBD2 and the histone deacetylase core of NuRD. This model is remarkably consistent with our previous studies, including the current paramagnetic relaxation enhancement data. The latter suggests that the free MBD2-IDR samples conformations similar to the bound structure. We tested this model of the complex extensively by mutating key contact residues and measuring binding using an intracellular bioluminescent resonance energy transfer assay. Furthermore, we identified protein contacts that, when mutated, disrupted gene silencing by NuRD in a cell model of fetal hemoglobin regulation. Hence, this work provides insights into the formation of NuRD and highlights critical binding pockets that may be targeted to block gene silencing for therapy. Importantly, we show that AlphaFold2 can generate a credible model of a large complex that involves an IDR that folds upon binding.

Keywords: AlphaFold2; DNA methylation; MBD2-NuRD; chromatin remodeling; intrinsically disordered protein.

Fig. 1.

Solvent PRE (sPRE) measurements highlight the structural propensity of the MBD2-IDR. (A) A cartoon diagram of MBD2 depicts the MBD and coiled-coil domains and the intrinsically disordered region (IDR). (B) A 2D ¹⁵N-HSQC spectra of WT and R286E/L287A mutant MBD2-IDR shows limited chemical shift dispersion in the ¹H dimension and sharp narrow resonances indicative of an unstructured domain. The R286E/L287A mutation leads to small chemical shift changes involving only a few resonances. (C) The sPRE rates are plotted per residue for the WT (blue squares) and R286E/L287A mutant (red squares) MBD2-IDR. For comparison, the difference between WT and mutant are plotted (open black squares) with R286 and L287 plotted as red circles and highlighted above with a red bar. Double-headed arrows indicate the three regions with a propensity to form alphahelices by chemical shift indexing. Error bars represent the uncertainty in the fit to six delay increments (±1 SD).

Fig. 2.

Site-specific labeling of the MBD2-IDR with a paramagnetic probe reveals long-range contacts. The ratio of 2D ¹⁵N-HSQC peak intensities between paramagnetic and diamagnetic states is plotted for the MBD2-IDR with site-specific labeling of S269 (A) or S316 (B) and for the MBD2-IDR double-mutant (R286E/L287A) with site-specific labeling of S269 (C) or S316 (D). The observed relaxation enhancement extends beyond sequentially adjacent residues indicating long-range transient contacts, which is most notable for the S316 label (B). The R286E/L287A double mutation (C and D) disrupts these transient contacts, as demonstrated by reduced relaxation enhancement of residues 290 to 300 for the S316 site-specific label (D). A vertical red bar indicates the position of the mutation. Vertical dashed lines break the figure into three regions as visual guides. Error bars represent the uncertainty in the ratio (±1 SD) from the error in the measurement of peak intensities.

Fig. 3.

Detecting MBD2-MTA2 complex formation in cells. (A) A diagram depicts the MTA2 and MBD2 constructs used in a NanoBRET assay to measure intracellular interaction. We fused the NL donor domain to either the N or C terminus of MTA2 and the HT acceptor domain to the N or C terminus of MBD2. (B and C) The BRET intensity is plotted for different donor and acceptor pairs, with the NL and HT domains on the N and C termini as indicated. The highest BRET intensity (red) was measured for constructs with the donor/acceptor on the C termini of the MTA2(ELM-SANT2) and the MDB2sc or IDRsc proteins. The R286E/L287A double mutation (gray) reduces the BRET intensity as compared to the WT protein. (D) We normalized the BRET intensity to the WT (1.0) and R286E/L287A mutation (0.0) of the IDRsc-MTA2(ELM-SANT) complex. Individual R286E and L287A mutations and truncations of the MBD2 IDR N terminus decrease the BRET intensity signal. Error bars represent ±1 SEM over four replicates. A P-value was calculated by an unpaired t test with Welch’s correction for each mutant in comparison with the wild type (n.s.: P > 0.05; *P < 0.05; **P < 0.001).

Fig. 4.

AlphaFold2 model of the MBD2-MTA2-HDAC1 complex. The AlphaFold2 model of MBD2sc bound to the MTA2(ELM-SANT)-HDAC1 dimer is shown as cartoon and surface representations, respectively. The MBD2sc is color-coded according to the linear diagram above, while MTA2(ELM-SANT) and HDAC1 surfaces are colored light and dark gray, respectively. Insets depict mixed cartoon and stick renderings that highlight specific contacts involving R246, V256, W283, and R286 residues of MBD2 (below, Left to Right), F362 of MBD2, and A157 and V160 of GATAD2A-CR1 (above, Left to Right) as described in the text. Individual residues are labeled for MBD2 (regular font), MTA2 or HDAC1 (italicized), or GATAD2A (italicized and underlined).

Fig. 5.

Mutating contact residues of the MBD2-IDR disrupts binding to MTA2. (A) Based on the AlphaFold2 model of the MBD2sc-MTA2-HDAC1 complex, we mutated contact residues and measured binding between IDRsc and MTA2 by NanoBRET. The measured BRET intensities were normalized to the WT (1.0) and R286/L286 mutant (0.0) signals to compare experiments. The mutations included residues within the MBD2-IDR 1st, 2nd, 3rd, and coiled-coil regions (regular font) and the GATAD2A CR1 domain (italicized and underlined). In addition, we tested combinations of the individual mutations (2X mutations), leading to a decrease in BRET intensity comparable to that of the R286E/L287A mutant. (B) The complementary mutation M220E in MTA2 (italicized) restores binding to the W283R mutation of MBD2. Furthermore, the I245R mutation of MBD2 provides additional favorable interaction with the second M220E of the MTA2 dimer, further increasing the NanoBRET signal. (C) Mixed cartoon and stick diagrams depict the favorable charge interactions introduced by these mutations. Error bars represent ±1 SEM over four replicates. A P-value was calculated by an unpaired t test with Welch’s correction for each mutant in comparison with the wild type (n.s.: P > 0.05; *P < 0.05; **P < 0.001).

Fig. 6.

Disrupting critical interactions between the MBD2-IDR and the HDCC abrogates gene silencing by the NuRD complex. (A) q-PCR results show the γ/(γ+β) mRNA ratio in parental HUDEP-2 cells, MBD2KO cells, and MBD2KO cells after add-back of either TAP-tag protein containing WT MBD2a or various MBD2a mutants. The error bars represent the mean ± SD of three independent biological repeats; *P < 0.05, **P < 0.001, ***P < 0.0001, and n.s. P > 0.05. (B) An immunoblot assay shows the total MBD2 protein level (as detected by anti-MBD2 antibody) and TAP-tagged MBD2 protein level (as determined by anti-Flag antibody) in individual control and add-back cells, as indicated.