Post-translational modification with one of the isoforms of the small ubiquitin-like modifier (SUMO) affects thousands of proteins in the human proteome. The binding of SUMO to SUMO interacting motifs (SIMs) can translate the SUMOylation event into functional consequences. The E3 ubiquitin ligase RNF4 contains multiple SIMs and connects SUMOylation to the ubiquitin pathway. SIM2 and SIM3 of RNF4 were shown to be the most important motifs to recognize SUMO chains. However, the study of SIM-SUMO complexes is complicated by their typically low affinity and variable binding of the SIMs in parallel and antiparallel orientations. We investigated properties of complexes formed by SUMO3 with peptides containing either SIM2 or SIM3 using molecular dynamics simulations. The affinities of the complexes were determined using a state-of-the-art free energy protocol and were found to be in good agreement with experimental data, thus corroborating our method. Long unrestrained simulations allowed a new interpretation of experimental results regarding the structure of the SIM-SUMO interface. We show that both SIM2 and SIM3 bind SUMO3 in parallel and antiparallel orientations and identified main interaction sites for acidic residues flanking the SIM. We noticed unusual SIM-SUMO interfaces in a previously reported NMR structure (PDB: 2mp2) of a complex formed by a SUMO3 dimer with the bivalent SIM2-SIM3 peptide. Computational determination of the individual SIM-SUMO affinities based on these structural arrangements yielded significantly higher dissociation constants. To our knowledge, our approach adds new opportunities to characterize individual SIM-SUMO complexes and suggests that further studies will be necessary to understand these interactions when occurring in multivalent form.

中文翻译：

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从 MD 模拟洞察 RNF4-SIM-SUMO 复合物的微观结构

使用一种小泛素样修饰剂 (SUMO) 的同种型进行翻译后修饰会影响人类蛋白质组中的数千种蛋白质。SUMO 与 SUMO 交互基序 (SIM) 的结合可以将 SUMO 化事件转化为功能结果。E3 泛素连接酶 RNF4 包含多个 SIM 并将 SUMOylation 连接到泛素途径。RNF4 的 SIM2 和 SIM3 被证明是识别 SUMO 链的最重要的基序。然而，SIM-SUMO 复合物的研究因其通常低亲和力和 SIM 平行和反平行方向的可变结合而变得复杂。我们使用分子动力学模拟研究了由 SUMO3 与含有 SIM2 或 SIM3 的肽形成的复合物的性质。复合物的亲和力是使用最先进的自由能协议确定的，并且发现与实验数据非常吻合，从而证实了我们的方法。长时间不受限制的模拟允许对有关 SIM-SUMO 界面结构的实验结果进行新的解释。我们表明 SIM2 和 SIM3 都以平行和反平行的方向结合 SUMO3，并确定了 SIM 侧翼酸性残基的主要相互作用位点。我们在先前报道的由 SUMO3 二聚体与二价 SIM2-SIM3 肽形成的复合物的 NMR 结构（PDB：2mp2）中注意到不寻常的 SIM-SUMO 界面。基于这些结构排列的单个 SIM-SUMO 亲和力的计算确定产生了显着更高的解离常数。据我们所知，