Kung Camy C-H, Naik Mandar T, Wang Szu-Huan, Shih Hsiu-Ming, Chang Che-Chang, Lin Li-Ying, Chen Chia-Lin, Ma Che, Chang Chi-Fon, Huang Tai-Huang
*Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.
§Scientific Instrument Center, Academia Sinica, Taipei 11529, Taiwan.
Biochem J. 2014 Aug 15;462(1):53-65. doi: 10.1042/BJ20140521.
The E3 ubiquitin ligase RNF4 (RING finger protein 4) contains four tandem SIM [SUMO (small ubiquitin-like modifier)-interaction motif] repeats for selective interaction with poly-SUMO-modified proteins, which it targets for degradation. We employed a multi-faceted approach to characterize the structure of the RNF4-SIMs domain and the tetra-SUMO2 chain to elucidate the interaction between them. In solution, the SIM domain was intrinsically disordered and the linkers of the tetra-SUMO2 were highly flexible. Individual SIMs of the RNF4-SIMs domains bind to SUMO2 in the groove between the β2-strand and the α1-helix parallel to the β2-strand. SIM2 and SIM3 bound to SUMO with a high affinity and together constituted the recognition module necessary for SUMO binding. SIM4 alone bound to SUMO with low affinity; however, its contribution to tetra-SUMO2 binding avidity is comparable with that of SIM3 when in the RNF4-SIMs domain. The SAXS data of the tetra-SUMO2-RNF4-SIMs domain complex indicate that it exists as an ordered structure. The HADDOCK model showed that the tandem RNF4-SIMs domain bound antiparallel to the tetra-SUMO2 chain orientation and wrapped around the SUMO protamers in a superhelical turn without imposing steric hindrance on either molecule.
E3泛素连接酶RNF4（环指蛋白4）包含四个串联的SIM[小泛素样修饰物（SUMO）相互作用基序]重复序列，用于与多聚SUMO修饰的蛋白质进行选择性相互作用，并将其作为降解靶点。我们采用了多方面的方法来表征RNF4-SIMs结构域和四聚SUMO2链的结构，以阐明它们之间的相互作用。在溶液中，SIM结构域本质上是无序的，四聚SUMO2的连接子具有高度的灵活性。RNF4-SIMs结构域的单个SIM在β2链和与β2链平行的α1螺旋之间的凹槽中与SUMO2结合。SIM2和SIM3与SUMO具有高亲和力结合，共同构成了SUMO结合所需的识别模块。单独的SIM4与SUMO结合亲和力较低；然而，当位于RNF4-SIMs结构域中时，它对四聚SUMO2结合亲和力的贡献与SIM3相当。四聚SUMO2-RNF4-SIMs结构域复合物的小角X射线散射（SAXS）数据表明它以有序结构存在。HADDOCK模型显示，串联的RNF4-SIMs结构域与四聚SUMO2链的方向反平行结合，并以超螺旋形式围绕SUMO原体缠绕，而不会对任何一个分子造成空间位阻。
