FC0087
Methyl-CpG binding domain protein 2 (MBD2) of nucleosome remodeling and deacytelase complex (NurD) (NurD MBD2)  -  DNA

Biological function
MBD2 binds densely methylated CpG islands and represses transcription of the associated genes through recruitment of the Nucleosome Remodeling and Deacetylase (NuRD) co-repressor complex. The methyl-CpG binding domain (MBD) family proteins recognize this methylated mark and repress the associated genes by recruiting different co-repressor complexes.

Domain organization/sequence features
N-terminal glycine-arginine repeat region (GR, 1-149 AA); a methyl-binding domain (MBD, 150-214 AA), which binds in vivo to densely methylated DNA; an uncharacterized domain of MBD2 (MBD2_IDR, 215-360AA) and a coiled-coil domain (CC, 361-393). C-terminal coiled-coil of MBD2 binds to the p66α component of NuRD, which contributes to the recruitment of CHD4 and gene silencing. This interaction in vivo relieves MBD2-mediated repression of target genes such as the embryonic and fetal α-type globin genes in adult erythroid cell culture systems. MBD2_IDR increases the overall binding affinity of MBD2 for methylated DNA. MBD2_IDR also recruits the histone deacetylase core components (RbAp48, HDAC2 and MTA2) of NuRD through a critical contact region requiring two contiguous amino acid residues, Arg²⁸⁶ and Leu²⁸⁷. Mutating these residues abrogates interaction of MBD2 with the histone deacetylase core and impairs the ability of MBD2 to repress the methylated tumor suppressor gene PRSS8 in MDA- MB-435 breast cancer cells.

Structural evidence
2D ¹⁵N-HSQC spectrum of the MBD2FLsc protein (150-393 AA, containing MBD_IDR and CC domains) bound to methylated DNA contains a few broadened and dispersed as well as many sharp and highly degenerate resonances. Plotting ¹ H,¹⁵ N chemical shift differences between resonances from the isolated MBD2_IDR and the same region in the MBD2FLsc:DNA confirms that the chemical shifts are very similar throughout the region (Δ < 0.05ppm). These findings show that the MBD2_IDR region remains largely disordered even in the context of full-length protein and does not appear to affect the structure or DNA binding mode of the MBD.

Biochemical evidence
The isolated MBD shows rapid on and off-rates, which requires steady state analysis to determine the overall binding affinity (K_D = 330 nM). The MBD2FLsc (150-393 AA) shows an ∼100-fold increase in affinity (K_D = 2 nM) as compared to the structured MBD2_MBD (K_D=330 nM). Mutations of two highly conserved residues (Arg 286, Leu 287) diminishes interactions with the histone deacetylase core complex, albeit these residues are located in the central portion, which might adopt a stable α-helical structure upon binding.

Structure/Mechanism
Evolutionary conserved Arg and Lys residues of the fuzzy region serve as nonspecific anchor to DNA.

Mechanism category
tethering

Isoforms, context-dependence
MBD2 alternative splice variants revealed that dominant isoform in embryonic stem cells, MBD2c, lacks most of the C-terminus including the MBD2_IDR and the coiled-coil domains. Consistent with the role of these two domains in recruiting CHD4 and the histone deacetylase core complex, MBD2c fails to interact with any of the NuRD components and lacks repressive activity.

Significance
Fuzziness enables a dynamic architecture of the NuRD complex wherein the subunits carrying the chromatin remodeling enzymatic activities unique to the NuRD complex can be mapped to two separate domains of MBD2: the MBD2_IDR. Such a segmental organization enables to simultaneously recruit the histone deacetylase core components and the chromatin remodeling subunit CHD4 through its interaction with p66α.

Submitted by
David C. Williams   david_willjr@med.unc.edu