Les of biological assembly of ubiquitin E3s. Examples of Ubiquitin E3s Name RING E3 Biological Assembly Monomer Homodimer Heterodimer Homodimer, heterodimer, or oligomers Element of multi-subunit (U-box) Monomer Homodimer HECT E3 RBR PCAF_N Atypical Monomer or oligomer Monomer or oligomer Monomer or component of multi-subunit Monomer Heterodimer Component of multi-subunit Protein (PDB ID) CBL (1fb), RNF38 (4v3l), CNOT4(1ur6), ARK2C (5d0m, 5d0k) RNF4 (4ppe, 4ap4), cIAP2(3eb6), BIRC7 (4auq),TRAF6 (3hcs, 5vo0) MDM2-MDMX (2vje), BRCA1-BARD1 (1jm7),RNF2-BMI1 (2ckl)TRAF6:TRAF5 (7l3l) TRIM household proteins (TRIM65(7jl2), TRIM5a(4tkp), TRIM28(6i9h), TRIM32(5fey)) APC/C (APC11 (4r2y, 5jg6, 5lt9)), CRL (RBX1 (4f52, 1ldk, 4a0l), RBX2 (7oni)) UBE4B generally known as UFD2 (2qj0) PRP19(2bay), CHIP (2c2v) SMURF1 (1zvd), NEDD4L (3jvz), WWPI (1nd7), E6AP(1c4z) Polmacoxib site PARKIN (5c23), HHARI (5tte), HOIP(4ljo) GCN5 (7by1) ZBF451(5d2m) ATG12-ATG5(4naw) RanBP2(1z5s)Some RING E3s have an extra ubiquitin-binding component that enhances enzymatic activity by stabilizing E2 ubiquitin [635]. For instance, the phosphate moiety of phosphor-Tyr36 of CBL-L types a hydrogen bond together with the Thr9 of ubiquitin, and loops adjacent for the RING domain of RNF38 speak to the Thr9-containing surface of ubiquitin. In ARK2C, RING E3 is expected for two ubiquitin-binding to exert transfer activity; a single ubiquitin is positioned around the same surface with the E2-binding surface and one more one particular binds for the opposite surface of RING E3 [66]. Inside the dimeric RING E3s, RNF4 and BIRC7, two domains cooperatively recognize ubiquitin: a single subunit and a C-terminal tail of a different subunit interact using the Gly35-containing surface of ubiquitin. On the other hand, TRIM25 makes use of a unique interface for ubiquitin recognition: the TRIM25 UBE2D1 (UbcH5a) ubiquitin complex structure revealed that the N-terminal helix of one particular subunit and C-terminal helix of a different subunit make a hydrophilic interaction together with the Gly35containing surface of ubiquitin. In addition to E2-E3 interactions, quite a few E3s harbor an more domain for interacting with all the backside surface of E2 that enhances the RING E3-E2 affinity but impacts activity disparately [670]. Some E2s, such as RAD6 and also the UbcH5, bind to ubiquitin around the backside surface of E2 to promote processive polyubiquitin chain formation [69,71,72]. These pieces of evidence indicated that additional elements, domains, and molecules have distinct roles. Additional structural and biochemical studies like these molecules are essential for understanding RING E3-mediated ubiquitylation [55]. 3.3.3. HECT There are 28 HECT E3s in humans [73]. The HECT E3s consist of an N-terminal substrate-binding domain along with a C-terminal HECT domain. C-terminal HECT is actually a domain consisting of 350 amino acids (Figure 3A). It was initial described in human papillomavirus (HPV) E6-associated protein (E6AP) five [73]. HECT E3s are Mouse Epigenetic Reader Domain divided into 3 groups determined by their N-terminal domain: NEDD4 family members, HERC family members, and HECTs with other proteinprotein interaction domains. The HECT domain itself is divided into two lobes which can be connected by a flexible hinge loop. The N-terminal lobe (N-lobe) binds to E2 ubiquitin,Molecules 2021, 26,eight ofand the C-terminal lobe (C-lobe) has the catalytic cysteine residue [74]. The flexible hinge enables the lobes to rotate, top to ubiquitin transfer reaction [75]. After the binding of E2 ubiquitin to the N-lobe, ubiquitin is transferred from E2 towards the catalytic cysteine o.