LIG_UBA3_1

Ubiquitination is a post-translational modification, which leads to the degradation of a substrate protein at the 26S proteasome. The conjugation of a Ubiquitin to its substrate involves a three-step multi-enzymatic process using an E1-(activating), E2-(conjugating) and E3-(ligase) enzyme (Brown,2015). One of the two main classes of E3 ubiquitin ligases contains a RING domain (either RBX1 or RBX2) that associates with a cullin subunit, which is why they are termed cullin/RING ligases (CRLs) (Petroski,2005).
CRLs are activated by covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal lysine. This process is called neddylation and prevents the association of a CRL with its inhibitor CAND1 (cullin-associated NEDD8-dissociated 1).
Analogous to ubiquitination, neddylation also involves a three-step enzymatic process. The NEDD8 E1 is a heterodimer composed of NAE1 and UBA3. NAE1-UBA3 interacts with two known NEDD E2s, UBE2M and UBE2F. NEDD8 E3 ligases are represented by the RING domains of CRLs. Cullins bind substrate recognition subunits, the RING domains acts as ligases by binding the E2~NEDD8 intermediate to catalyse NEDD8 transfer. UBE2M preferentially interacts with the E3 RBX1 and UBE2F with RBX2 (Brown,2015). RBX1 can interact with CUL1-4, whereas RBX2 exclusively interacts with CUL5, leading to UBE2F specificity for CUL5 (Monda,2013).
Defective in cullin neddylation protein 1-like proteins 1-5 (DCNL1-5), are scaffold-like co-E3s that regulate cullin neddylation by acting as cofactors for RBX1 and RBX2.
All essential functions of DCNLs for neddylation reside within the PONY domain. DCNLs bind an E2~NEDD8 intermediate via their PONY domain and mediate the recruitment of an E3 cullin subunit through binding to its WHB subdomain (Kurz,2008). Also, the PONY domain contains the binding site for the UBE2M and UBE2F N-terminal motifs, based on a conserved ^M[IL].L core sequence. These peptides bind into a deep conserved hydrophobic pocket of DCNLs PONY domain (Monda,2013, 3BQ3, 3TDU, 4GAO, 4GBA). The binding motifs are overlapping, with the motifs responsible for NEDD8 E2~E1 interactions (LIG_UBA3_1), in fact in the case of UBE2F the motif residues are identical. A motif characterised by a conserved ϕ-ϕ-x-ϕ pattern can be found in UBE2M and UBE2f across species (ϕ, hydrophobic residue; x, any residue).
The NAE1-UBA3 is required to be able to recognise both E2s which is in partly mediated by a distinct binding of the E2s core domain to the E1 ubiquitin fold domain and partly by a E2 N-terminal binding motif which binds to a groove of UBA3 in the E1 complex and selects for the NEDD8 pathway. Multiple E1-E2 interaction surfaces may serve in increasing affinity, while remaining adaptable to both E2 but at the same time preventing any mischarges of the wrong E2 (9250909, 3FN1, 1TT5). It was proven, that both the E2 N-terminal binding peptide and the E2 core domain must bind UBA3 simultaneously for optimal transfer of NEDD8 from E1 to E2. The interaction between NAE1-UBA3 and the E2 N-terminal peptides are probably unique to the NEDD8 pathway, because many E2s from other UBLs lack N-terminal extensions. Because the N-terminal extention of UBE2M was shown to play an important role in mitogenisis in CSF-1 dependent proliferation, NAE1-UBA3 and the E2s may serve as good targets for agents with antimitotic effects (Huang,2004).