Biochemistry. 2019 Feb 12;58(6):621-632. doi: 10.1021/acs.biochem.8b01045. Epub 2019 Jan 10.
SUMO, a conserved ubiquitin-like protein, is conjugated to a multitude of cellular proteins to maintain genomic integrity and resist genotoxic stress. Studies of the SUMO E2 conjugating enzyme mutant, UBC9, suggested that altered substrate specificity enhances cell sensitivity to DNA damaging agents. Using nuclear magnetic resonance chemical shift studies, we confirm that the mutation does not alter the core globular fold of UBC9, while N relaxation measurements demonstrate mutant-induced stabilization of distinct chemical states in residues near the active site cysteine and substrate recognition motifs. We further demonstrate that the P123L substitution induces a switch from the preferential addition of SUMO to lysine residues in unstructured sites to acceptor lysines embedded in secondary structures, thereby also inducing alterations in SUMO chain linkages. Our results provide new insights regarding the impact that structural dynamics of UBC9 have on substrate selection and specifically SUMO chain formation. These findings highlight the potential contribution of nonconsensus SUMO targets and/or alternative SUMO chain linkages on DNA damage response and chemotherapeutic sensitivity.
SUMO 是一种保守的泛素样蛋白，可与多种细胞蛋白结合，以维持基因组完整性并抵抗遗传毒性应激。SUMO E2 连接酶突变体 UBC9 的研究表明，改变底物特异性会增强细胞对 DNA 损伤剂的敏感性。通过核磁共振化学位移研究，我们证实该突变不会改变 UBC9 的核心球状折叠，而 N 弛豫测量则证明了突变诱导了活性位点半胱氨酸和底物识别模体附近的不同化学状态的稳定。我们进一步证明，P123L 取代诱导了从优先将 SUMO 添加到无规则结构位点的赖氨酸残基到嵌入二级结构中的接受体赖氨酸的转变，从而也诱导了 SUMO 链连接的改变。我们的结果提供了有关 UBC9 结构动力学对底物选择和特定 SUMO 链形成的影响的新见解。这些发现强调了非共识 SUMO 靶标和/或替代 SUMO 链连接在 DNA 损伤反应和化学敏感性方面的潜在贡献。
