Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore, 117558, Singapore.
Department of Biochemistry, National University of Singapore, 28 Medical Drive, Singapore, 117546, Singapore.
Adv Sci (Weinh). 2023 Sep;10(27):e2207672. doi: 10.1002/advs.202207672. Epub 2023 Aug 3.
HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.
HACE1 是一种含有锚蛋白重复（AKR）的 HECT 型 E3 泛素连接酶，可与多种底物相互作用并泛素化。虽然 HACE1 是一种众所周知的肿瘤抑制因子，但它的结构和泛素化模式尚不清楚。作者展示了人源 HACE1 的冷冻电镜结构以及体外功能研究，这些研究提供了有关 HACE1 酶活性如何受到调节的深入了解。HACE1 由 N 端 AKR 结构域、中间（MID）结构域和 C 端 HECT 结构域组成。其独特的 G 形结构作为同源二聚体相互作用，单体以反平行方式排列。在这种二聚体排列中，HACE1 的泛素化活性受到阻碍，因为一个单体的 N 端螺旋限制了另一个单体 C 端结构域的进入。体外泛素化测定、氢氘交换质谱（HDX-MS）分析、突变和计算建模表明，HACE1 的 MID 结构域与 AKRs 一起在 RAC1 底物识别中发挥关键作用。
