Ubiquitin ligases (E3s) are pivotal specificity determinants in the ubiquitin system by selecting substrates and decorating them with distinct ubiquitin signals. However, structure determination of the underlying, specific E3-substrate complexes has proven challenging owing to their transient nature. In particular, it is incompletely understood how members of the catalytic cysteine-driven class of HECT-type ligases (HECTs) position substrate proteins for modification. Here, we report a cryogenic electron microscopy (cryo-EM) structure of the full-length human HECT HACE1, along with solution-based conformational analyses by small-angle X-ray scattering and hydrogen–deuterium exchange mass spectrometry. Structure-based functional analyses in vitro and in cells reveal that the activity of HACE1 is stringently regulated by dimerization-induced autoinhibition. The inhibition occurs at the first step of the catalytic cycle and is thus substrate-independent. We use mechanism-based chemical crosslinking to reconstitute a complex of activated, monomeric HACE1 with its major substrate, RAC1, determine its structure by cryo-EM and validate the binding mode by solution-based analyses. Our findings explain how HACE1 achieves selectivity in ubiquitinating the active, GTP-loaded state of RAC1 and establish a framework for interpreting mutational alterations of the HACE1–RAC1 interplay in disease. More broadly, this work illuminates central unexplored aspects in the architecture, conformational dynamics, regulation and specificity of full-length HECTs.

泛素连接酶 (E3) 通过选择底物并用不同的泛素信号修饰它们，成为泛素系统中关键的特异性决定因素。然而，由于其瞬态性质，底层特定 E3 底物复合物的结构确定具有挑战性。特别是，催化半胱氨酸驱动的 HECT 型连接酶 (HECT) 类型的成员如何定位底物蛋白进行修饰尚不完全清楚。在这里，我们报告了全长人 HECT HACE1 的低温电子显微镜 (cryo-EM) 结构，以及通过小角 X 射线散射和氢氘交换质谱进行的基于溶液的构象分析。体外和细胞内基于结构的功能分析表明，HACE1 的活性受到二聚化诱导的自抑制的严格调节。这种抑制发生在催化循环的第一步，因此与底物无关。我们使用基于机制的化学交联来重建活化的单体 HACE1 及其主要底物 RAC1 的复合物，通过冷冻电镜确定其结构，并通过基于溶液的分析验证结合模式。我们的研究结果解释了 HACE1 如何实现选择性泛素化 RAC1 的活性、GTP 负载状态，并建立了一个解释疾病中 HACE1-RAC1 相互作用的突变改变的框架。更广泛地说，这项工作阐明了全长 HECT 的架构、构象动力学、调节和特异性方面未探索的核心方面。