To enable the efficient and specific transfer of ubiquitin to diverse target proteins and ubiquitin chain formation, ubiquitylation enzymes rely on intricate conformational changes. In particular, catalytic cysteine-dependent ubiquitin ligases sequentially catalyze distinct chemical reactions, which implicitly requires conformational dynamics. In line with this concept, RBR, RCR, and HECT-type ligases — while structurally different from each other — all contain catalytic domains with built-in flexibility that have been crystallized in various states, providing snapshots of their conformational trajectories. However, which mechanisms trigger the underlying transitions and lend directionality to the conformational cycle of cysteine-dependent ligases is incompletely understood. It is also largely unknown whether disease-causing mutations in the respective ligase genes alter the conformational cycling of the enzymes to perturb their catalytic functions and/or regulation. We address these key questions for selected members of the HECT ligase family. Our results are expected to facilitate an efficient therapeutic targeting of HECT ligases by small molecules, which has proven extraordinarily challenging.