Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Budapest, 1111, Hungary.
Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, 1117, Hungary.
Sci Rep. 2018 Mar 12;8(1):4326. doi: 10.1038/s41598-018-22145-8.
Human deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), essential for DNA integrity, acts as a survival factor for tumor cells and is a target for cancer chemotherapy. Here we report that the Staphylococcal repressor protein Stl (Stl) forms strong complex with human dUTPase. Functional analysis reveals that this interaction results in significant reduction of both dUTPase enzymatic activity and DNA binding capability of Stl. We conducted structural studies to understand the mechanism of this mutual inhibition. Small-angle X-ray scattering (SAXS) complemented with hydrogen-deuterium exchange mass spectrometry (HDX-MS) data allowed us to obtain 3D structural models comprising a trimeric dUTPase complexed with separate Stl monomers. These models thus reveal that upon dUTPase-Stl complex formation the functional homodimer of Stl repressor dissociates, which abolishes the DNA binding ability of the protein. Active site forming dUTPase segments were directly identified to be involved in the dUTPase-Stl interaction by HDX-MS, explaining the loss of dUTPase activity upon complexation. Our results provide key novel structural insights that pave the way for further applications of the first potent proteinaceous inhibitor of human dUTPase.
人脱氧尿苷 5'-三磷酸核苷水解酶（dUTPase）对 DNA 完整性至关重要，它是肿瘤细胞的生存因子，也是癌症化疗的靶标。在这里，我们报告葡萄球菌阻遏蛋白 Stl（Stl）与人 dUTPase 形成强复合物。功能分析表明，这种相互作用导致 dUTPase 的酶活性和 Stl 的 DNA 结合能力显著降低。我们进行了结构研究以了解这种相互抑制的机制。小角 X 射线散射（SAXS）辅以氘-氢交换质谱（HDX-MS）数据使我们能够获得包含与单独的 Stl 单体复合的三聚体 dUTPase 复合物的 3D 结构模型。这些模型表明，在 dUTPase-Stl 复合物形成时，阻遏蛋白的功能性同源二聚体解离，从而使该蛋白丧失 DNA 结合能力。通过 HDX-MS 直接鉴定出形成活性位点的 dUTPase 片段参与 dUTPase-Stl 相互作用，解释了复合物形成时 dUTPase 活性的丧失。我们的研究结果提供了关键的新结构见解，为进一步应用人类 dUTPase 的首个有效的蛋白质抑制剂铺平了道路。
