SMAD transcription factors, the main effectors of the TGFβ (transforming growth factor β) network, have a mixed architecture of globular domains and flexible linkers. Such a complicated architecture precluded the description of their full-length (FL) structure for many years. In this study, we unravel the structures of SMAD4 and SMAD2 proteins through an integrative approach combining Small-angle X-ray scattering, Nuclear Magnetic Resonance spectroscopy, X-ray, and computational modeling. We show that both proteins populate ensembles of conformations, with the globular domains tethered by disordered and flexible linkers, which defines a new dimension of regulation. The flexibility of the linkers facilitates DNA and protein binding and modulates the protein structure. Yet, SMAD4FL is monomeric, whereas SMAD2FL is in different monomer-dimer-trimer states, driven by interactions of the MH2 domains. Dimers are present regardless of the SMAD2FL activation state and concentration. Finally, we propose that SMAD2FL dimers are key building blocks for the quaternary structures of SMAD complexes.

SMAD 转录因子是 TGFβ（转化生长因子 β）网络的主要效应器，具有球状结构域和灵活接头的混合结构。如此复杂的架构多年来一直无法描述其全长（FL）结构。在这项研究中，我们通过结合小角 X 射线散射、核磁共振波谱、X 射线和计算模型的综合方法揭示了 SMAD4 和 SMAD2 蛋白的结构。我们表明，这两种蛋白质都填充了构象集合，球状结构域由无序且灵活的连接体束缚，这定义了调控的新维度。连接子的灵活性有利于 DNA 和蛋白质的结合并调节蛋白质结构。然而，SMAD4FL 是单体，而 SMAD2FL 处于不同的单体-二聚体-三聚体状态，由 MH2 结构域的相互作用驱动。无论 SMAD2FL 激活状态和浓度如何，二聚体都存在。最后，我们提出 SMAD2FL 二聚体是 SMAD 复合物四级结构的关键构建模块。