School of Life Sciences, Tianjin University, Tianjin 300072, China.
Department of Molecular Biology, Ariel University, Ariel 40700, Israel.
J Biol Chem. 2019 Oct 25;294(43):15733-15742. doi: 10.1074/jbc.RA119.010123. Epub 2019 Sep 4.
REV7, also termed mitotic arrest-deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7-RAN and REV7-IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00-2.35 Å resolutions. The structures revealed that both RAN and IpaB fragments bind the "safety belt" region of REV7, inducing rearrangement of the C-terminal β-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity.
REV7，也称为有丝分裂阻滞缺陷 2 样 2（MAD2L2 或 MAD2B），作为广泛的细胞途径中的相互作用模块，包括跨损伤 DNA 合成、细胞周期控制和非同源末端连接。已经鉴定出许多 REV7 结合伴侣，包括人类小 GTP 酶 Ras 相关核蛋白（RAN），它作为 REV7 的潜在上游调节剂。值得注意的是，侵袭素 IpaB 劫持 REV7 以破坏细胞周期控制，以防止肠上皮细胞更新并促进细菌定植。然而，REV7-RAN 和 REV7-IpaB 相互作用的结构细节在很大程度上尚不清楚。在这里，我们使用融合蛋白和刚性麦芽糖结合蛋白标记策略，以 2.00-2.35 Å 的分辨率确定了这两个复合物的晶体结构。这些结构表明，RAN 和 IpaB 片段都结合 REV7 的“安全带”区域，诱导 REV7 的 C 末端β-片层区域的重排，该区域在 REV7 相关复合物中保守。值得注意的是，尽管 RAN 和 IpaB 的 REV7 结合基序都与 REV7 具有一些独特的相互作用，但它们都共享保守残基。结构比对表明，REV7 在安全带区域内具有一个衔接区域，该区域可以重排二级结构以适应各种不同的配体。我们的结构和生化结果进一步表明，REV7 优先结合 GTP 结合的 RAN，这表明 RAN 的 GTP/GDP 结合转变可能作为控制 REV7 活性的分子开关。这些结果提供了对 REV7 在细胞周期控制中的调节机制的深入了解，这可能有助于开发针对 REV7 活性的小分子抑制剂。
