Kobilka Institute of Innovative Drug Discovery, School of Medicine, The Chinese University of Hong Kong, Shenzhen, Shenzhen, 518172, China.
Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen, 518055, China.
Nat Commun. 2023 Nov 22;14(1):7631. doi: 10.1038/s41467-023-43174-6.
AMBRA1 is a tumor suppressor protein that functions as a substrate receptor of the ubiquitin conjugation system with roles in autophagy and the cell cycle regulatory network. The intrinsic disorder of AMBRA1 has thus far precluded its structural determination. To solve this problem, we analyzed the dynamics of AMBRA1 using hydrogen deuterium exchange mass spectrometry (HDX-MS). The HDX results indicated that AMBRA1 is a highly flexible protein and can be stabilized upon interaction with DDB1, the adaptor of the Cullin4A/B E3 ligase. Here, we present the cryo-EM structure of AMBRA1 in complex with DDB1 at 3.08 Å resolution. The structure shows that parts of the N- and C-terminal structural regions in AMBRA1 fold together into the highly dynamic WD40 domain and reveals how DDB1 engages with AMBRA1 to create a binding scaffold for substrate recruitment. The N-terminal helix-loop-helix motif and WD40 domain of AMBRA1 associate with the double-propeller fold of DDB1. We also demonstrate that DDB1 binding-defective AMBRA1 mutants prevent ubiquitination of the substrate Cyclin D1 in vitro and increase cell cycle progression. Together, these results provide structural insights into the AMBRA1-ubiquitin ligase complex and suggest a mechanism by which AMBRA1 acts as a hub involved in various physiological processes.
AMBRA1 是一种肿瘤抑制蛋白，作为泛素连接系统的底物受体发挥作用，在自噬和细胞周期调控网络中具有重要作用。AMBRA1 的固有无序性迄今为止阻止了其结构的确定。为了解决这个问题，我们使用氢氘交换质谱（HDX-MS）分析了 AMBRA1 的动力学。HDX 结果表明，AMBRA1 是一种高度灵活的蛋白质，与 DDB1 相互作用后可以稳定下来，DDB1 是 Cullin4A/B E3 连接酶的衔接蛋白。在这里，我们呈现了 AMBRA1 与 DDB1 复合物的冷冻电镜结构，分辨率为 3.08 Å。该结构表明，AMBRA1 的 N-和 C-末端结构区域的部分折叠成高度动态的 WD40 结构域，并揭示了 DDB1 如何与 AMBRA1 相互作用，为底物募集创建结合支架。AMBRA1 的 N-端螺旋环螺旋基序和 WD40 结构域与 DDB1 的双推进器折叠相关。我们还证明，DDB1 结合缺陷型 AMBRA1 突变体可阻止体外底物 Cyclin D1 的泛素化，并增加细胞周期进程。总之，这些结果为 AMBRA1-泛素连接酶复合物提供了结构见解，并提出了 AMBRA1 作为参与各种生理过程的枢纽的作用机制。
