Institut für Physikalische Chemie, Westfälische Wilhelms-Universität Münster, Münster, Germany; Center for Multiscale Theory and Computation, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Münster, Germany.
Biophys J. 2020 Oct 20;119(8):1558-1567. doi: 10.1016/j.bpj.2020.09.003. Epub 2020 Sep 11.
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.
用小泛素样修饰物（SUMO）的一种同工型进行的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相互作用基序（SIM）的结合可将SUMO化事件转化为功能后果。E3泛素连接酶RNF4包含多个SIM，并将SUMO化与泛素途径相连。RNF4的SIM2和SIM3被证明是识别SUMO链的最重要基序。然而，SIM-SUMO复合物的研究因它们通常较低的亲和力以及SIM以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SUMO3与含有SIM2或SIM3的肽形成的复合物的性质。使用最先进的自由能协议确定了复合物的亲和力，发现其与实验数据高度一致，从而证实了我们的方法。长时间的无约束模拟对关于SIM-SUMO界面结构的实验结果给出了新的解释。我们表明，SIM2和SIM3均以平行和反平行方向结合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）中注意到了不寻常的SIM-SUMO界面。基于这些结构排列对单个SIM-SUMO亲和力进行的计算测定产生了明显更高的解离常数。据我们所知，我们的方法为表征单个SIM-SUMO复合物提供了新的机会，并表明当以多价形式发生这些相互作用时，需要进一步研究以了解它们。
