Integrative Life Sciences Program, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
J Biol Chem. 2013 Feb 1;288(5):3419-27. doi: 10.1074/jbc.M112.431346. Epub 2012 Dec 13.
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.
甲基胞嘧啶结合域 2（MBD2）-核小体重塑和去乙酰化酶（NuRD）复合物识别甲基化 DNA，并通过组蛋白去乙酰化酶和核小体重塑功能沉默相关基因的表达。我们之前的结构研究表明，MBD2 与包含 GATA 锌指结构域 2A（GATAD2A/p66α）的蛋白质之间的卷曲螺旋相互作用招募染色质解旋酶 DNA 结合蛋白（CHD4/Mi2β）到 NuRD 复合物中，并且对于 MBD2 介导的体内 DNA 甲基化依赖性基因沉默是必要的（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.，和 Williams，D. C.，Jr.（2011）p66α-MBD2 卷曲螺旋相互作用和 Mi-2 的募集对于 MBD2-NuRD 复合物的珠蛋白基因沉默至关重要。美国国家科学院院刊 108,7487-7492）。与迄今为止研究的大多数卷曲螺旋不同，p66α-MBD2 相互作用形成两个肽之间的反平行异二聚体复合物，这两个肽在分离时主要是单体。为了进一步表征驱动异二聚体特异性和高亲和力结合的这种复合物的独特特征，我们对 MBD2 及其相关同源物 MBD3、MBD3 样蛋白 1（MBD3L1）和 MBD3 样蛋白 2（MBD3L2）以及修饰电荷-电荷相互作用和螺旋倾向的特定突变进行了生物物理分析卷曲螺旋结构域。分析超速离心分析表明，即使对于 MBD2，分离的单个肽在 300μM 的浓度下仍保持单体状态。圆二色性分析表明，卷曲螺旋结构域的螺旋含量与 p66α 的结合亲和力之间存在直接相关性。此外，每个肽的互补静电表面电势和固有螺旋含量对于维持高亲和力结合是必要的。这些因素导致 p66α 与不同 MBD2 同源物的结合亲和力层次结构（MBD2≈MBD3＞MBD3L1≈MBD3L2），并表明 NuRD 复合物在组织和生命周期阶段特异性沉默中的分级调节模型。
