Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic; National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic.
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic.
J Mol Biol. 2020 Jun 12;432(13):3820-3837. doi: 10.1016/j.jmb.2020.04.024. Epub 2020 May 8.
A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular processes ensuring proper cohesion, condensation and replication. They share a common SMC-kleisin architecture allowing them to embrace DNA. In SMC5/6, the NSE1 and NSE3 KITE and NSE4 kleisin subunits form a stable subcomplex that binds DNA and regulates essential processes. In addition, NSE5 and NSE6 subunits associate with the core SMC5/6 complex and recruit it to DNA repair sites. The architecture of the SMC5/6 complex is crucial for its proper functioning, and mutations within the human SMC5/6 subunits result in severe syndromes. Therefore, we aimed to analyze interactions within the human SMC5/6 complex and determine its detailed architecture. Firstly, we analyzed different parts of SMC5/6 by crosslinking and MS/MS analysis. Our data suggested domain arrangements of hNSE1-hNSE3 and orientation of hNSE4 within the hNSE1-hNSE3-hNSE4 subcomplex. The crosslinking and electron microscopic analysis of the SMC5/6 core complex showed its rod-like architecture with juxtaposed hSMC5-hSMC6 arms. Additionally, we observed fully or partially opened hSMC5-hSMC6 shapes with the hNSE1-hNSE3-hNSE4 trimer localized in the SMC head domains. To complete mapping of the human SMC5/6 complex architecture, we analyzed positions of hNSE5-hNSE6 at the hSMC5-hSMC6 arms. We showed that hNSE6 binding to hNSE5 and the coiled-coil arm of hSMC6 is mediated by a conserved FAM178 domain, which we therefore renamed CANIN (Coiled-coil SMC6 And NSE5 INteracting) domain. Interestingly, hNSE6 bound both hSMC5 and hSMC6 arms, suggesting that hNSE6 may lock the arms and regulate the dynamics of the human SMC5/6 complex.
一个结构维护染色体（SMC）复合物家族对于确保适当的凝聚、浓缩和复制的关键细胞过程是必不可少的。它们共享一个常见的 SMC-连接酶结构，允许它们拥抱 DNA。在 SMC5/6 中，NSE1 和 NSE3 KITE 和 NSE4 连接酶亚基形成一个稳定的亚复合物，与 DNA 结合并调节基本过程。此外，NSE5 和 NSE6 亚基与核心 SMC5/6 复合物结合，并将其招募到 DNA 修复位点。SMC5/6 复合物的结构对于其正常功能至关重要，人类 SMC5/6 亚基内的突变会导致严重的综合征。因此，我们旨在分析人类 SMC5/6 复合物内的相互作用，并确定其详细结构。首先，我们通过交联和 MS/MS 分析分析了 SMC5/6 的不同部分。我们的数据表明 hNSE1-hNSE3 和 hNSE4 内的结构域排列在 hNSE1-hNSE3-hNSE4 亚复合物内。SMC5/6 核心复合物的交联和电子显微镜分析显示其具有杆状结构，hSMC5-hSMC6 臂并列。此外，我们观察到 hSMC5-hSMC6 形状完全或部分打开，hNSE1-hNSE3-hNSE4 三聚体定位于 SMC 头部结构域。为了完成人类 SMC5/6 复合物结构的映射，我们分析了 hNSE5-hNSE6 在 hSMC5-hSMC6 臂上的位置。我们表明，hNSE6 与 hNSE5 和 hSMC6 的卷曲螺旋臂的结合是由一个保守的 FAM178 结构域介导的，我们因此将其重新命名为 CANIN（卷曲螺旋 SMC6 和 NSE5 相互作用）结构域。有趣的是，hNSE6 结合了 hSMC5 和 hSMC6 臂，这表明 hNSE6 可能锁定臂并调节人类 SMC5/6 复合物的动力学。
