There are hundreds of E3 ligases identified in mammalian ubiquitination cascades. Based on structural and functional differences, E3 ligases are subdivided into two major groups, the HECT family and the RING family. HECT E3 ligases catalyze a two step-reaction, starting with the formation of a thioester bond with ubiquitin followed by substrate recognition and ubiquitin transfer. The RING E3 ligases bind to ubiquitin-E2 heterodimer and protein substrates in a simultaneous manner through scaffold and adaptor proteins, facilitating an efficient transfer of ubiquitin onto specific substrates.

E3 ligases catalyze different architectures of ubiquitination. Some target proteins are ubiquitylated at one lysine residue only with one ubiquitin molecule (mono-ubiquitination) or with multiple ubiquitin molecules conjugated to each other (poly-ubiquitination). The polyubiquitin chain can form at the same (unbranched) or at different (branched) lysine residues in the ubiquitin sequence. Finally, E3 ligase substrates can be mono-ubiquitylated at multiple lysine residues (multi-ubiquitination).