Birkou Maria, Chasapis Christos T, Marousis Konstantinos D, Loutsidou Ariadni K, Bentrop Detlef, Lelli Moreno, Herrmann Torsten, Carthy Jonathon M, Episkopou Vasso, Spyroulias Georgios A
Department of Pharmacy, University of Patras, GR-26504 Patras, Greece.
Institute of Physiology II, Faculty of Medicine, University of Freiburg, 79104 Freiburg, Germany.
J Mol Biol. 2017 Jul 21;429(15):2373-2386. doi: 10.1016/j.jmb.2017.06.012. Epub 2017 Jun 22.
Arkadia (Rnf111) is an E3 ubiquitin ligase that plays a central role in the amplification of transforming growth factor beta (TGF-β) signaling responses by targeting for degradation the negative regulators of the pathway, Smad6 and Smad7, and the nuclear co-repressors Ski and Skil (SnoN). Arkadia's function in vivo depends on the really interesting new gene (RING)-H2 interaction with the E2 enzyme UbcH5b in order to ligate ubiquitin chains on its substrates. A conserved tryptophan (W972) in the C-terminal α-helix is widely accepted as essential for E2 recruitment and interaction and thus also for E3 enzymatic activity. The present NMR-driven study provides an atomic-level investigation of the structural and dynamical properties of two W972 Arkadia RING mutants, attempting to illuminate for the first time the differences between a functional and a nonfunctional mutant W972A and W972R, respectively. A TGF-β-responsive promoter driving luciferase was used to assay for Arkadia function in vivo. These experiments showed that the Arkadia W972A mutant has the same activity as wild-type (WT) Arkadia in enhancing TGF-β signaling responses, while W972R does not. Only minor structural differences exist between the W972A RING domain and WT-RING. In contrast, the W972R mutant hardly interacts with E2. The loss of function correlates with structural changes in the C-terminal α-helix and an increase in the distance between the Zn(II) ions. Our data show that the position occupied by W972 within WT Arkadia is critical for the function of RING and that it depends on the nature of the residue at this position.
阿卡迪亚（Rnf111）是一种E3泛素连接酶，通过靶向降解该信号通路的负调节因子Smad6和Smad7以及核共抑制因子Ski和Skil（SnoN），在转化生长因子β（TGF-β）信号反应的放大过程中发挥核心作用。阿卡迪亚在体内的功能依赖于其与E2酶UbcH5b的真核生物有趣新基因（RING）-H2相互作用，以便在其底物上连接泛素链。C端α螺旋中的保守色氨酸（W972）被广泛认为对于E2的招募和相互作用至关重要，因此对于E3酶活性也至关重要。目前这项由核磁共振驱动的研究对两个W972阿卡迪亚RING突变体的结构和动力学特性进行了原子水平的研究，试图首次阐明功能性和非功能性突变体W972A和W972R之间的差异。使用驱动荧光素酶的TGF-β反应性启动子来检测阿卡迪亚在体内的功能。这些实验表明，阿卡迪亚W972A突变体在增强TGF-β信号反应方面具有与野生型（WT）阿卡迪亚相同的活性，而W972R则没有。W972A RING结构域与WT-RING之间仅存在微小的结构差异。相比之下，W972R突变体几乎不与E2相互作用。功能丧失与C端α螺旋的结构变化以及锌（II）离子之间距离的增加有关。我们的数据表明，WT阿卡迪亚中W972所占据的位置对于RING的功能至关重要，并且这取决于该位置残基的性质。
