The methyl-cytosine binding domain 2 (MBD2)-nucleosome remodeling and 
deacetylase (NuRD) complex recognizes methylated DNA and silences expression of 
associated genes through histone deacetylase and nucleosome remodeling 
functions. Our previous structural work demonstrated that a coiled-coil 
interaction between MBD2 and GATA zinc finger domain containing 2A 
(GATAD2A/p66α) proteins recruits the chromodomain helicase DNA-binding protein 
(CHD4/Mi2β) to the NuRD complex and is necessary for MBD2-mediated DNA 
methylation-dependent gene silencing in vivo (Gnanapragasam, M. N., Scarsdale, 
J. N., Amaya, M. L., Webb, H. D., Desai, M. A., Walavalkar, N. M., Wang, S. Z., 
Zu Zhu, S., Ginder, G. D., and Williams, D. C., Jr. (2011) p66α-MBD2 coiled-coil 
interaction and recruitment of Mi-2 are critical for globin gene silencing by 
the MBD2-NuRD complex. Proc. Natl. Acad. Sci. U.S.A. 108, 7487-7492). The 
p66α-MBD2 interaction differs from most coiled-coils studied to date by forming 
an anti-parallel heterodimeric complex between two peptides that are largely 
monomeric in isolation. To further characterize unique features of this complex 
that drive heterodimeric specificity and high affinity binding, we carried out 
biophysical analyses of MBD2 and the related homologues MBD3, MBD3-like protein 
1 (MBD3L1), and MBD3-like protein 2 (MBD3L2) as well as specific mutations that 
modify charge-charge interactions and helical propensity of the coiled-coil 
domains. Analytical ultracentrifugation analyses show that the individual 
peptides remain monomeric in isolation even at 300 μM in concentration for MBD2. 
Circular dichroism analyses demonstrate a direct correlation between helical 
content of the coiled-coil domains in isolation and binding affinity for p66α. 
Furthermore, complementary electrostatic surface potentials and inherent helical 
content of each peptide are necessary to maintain high-affinity association. 
These factors lead to a binding affinity hierarchy of p66α for the different 
MBD2 homologues (MBD2 ≈ MBD3 > MBD3L1 ≈ MBD3L2) and suggest a hierarchical 
regulatory model in tissue and life cycle stage-specific silencing by NuRD 
complexes.