Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599.
Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298.
Proc Natl Acad Sci U S A. 2023 Aug 15;120(33):e2307287120. doi: 10.1073/pnas.2307287120. Epub 2023 Aug 8.
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.
核小体重塑和去乙酰化酶（NuRD）复合物改变核小体定位和染色质紧缩，以调节基因表达。甲基-CpG 结合域蛋白 2 和 3（MBD2 和 MBD3）在复合物形成中起着关键作用；然而，它们与其他 NuRD 成分相互作用的分子细节尚未完全阐明。我们之前表明，MBD2 的一个固有无序区域（IDR）是与 NuRD 的组蛋白去乙酰化酶核心结合所必需和充分的。在此基础上，我们使用溶剂和位点特异性顺磁弛豫增强测量来测量 MBD2-IDR 的固有结构倾向。然后，我们使用 AlphaFold2 机器学习软件生成 MBD2 与 NuRD 组蛋白去乙酰化酶核心之间复合物的模型。该模型与我们之前的研究非常一致，包括当前的顺磁弛豫增强数据。后者表明，游离的 MBD2-IDR 样品采取类似于结合结构的构象。我们通过突变关键接触残基并使用细胞内生物发光共振能量转移测定测量结合来广泛测试该复合物模型。此外，我们确定了蛋白质接触点，当这些接触点发生突变时，会破坏 NuRD 在胎儿血红蛋白调节的细胞模型中的基因沉默。因此，这项工作提供了对 NuRD 形成的深入了解，并突出了可能成为阻止基因沉默以进行治疗的关键结合口袋。重要的是，我们表明 AlphaFold2 可以生成一个涉及折叠后结合的 IDR 的大型复合物的可信模型。
