LIG_DCNL_PONY_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 contain a RING domain (either RBX1 or RBX2) which associates with a cullin subunit which is why they are termed cullin/RING ligases (CRLs) (Petroski,2005).
CRLs are activated by covalent attachment of 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, which regulate cullin neddylation by acting as cofactors for RBX1 and RBX2. The structure of DCNLs consists of an N-terminal ubiquitin associated domain (UBA domain) and a C-terminal potentiating neddylation domain (PONY domain), connected over a disordered linker.
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 a E3 cullin subunit through binding to its WHB subdomain (Kurz,2008). The PONY domain contains the binding site for the UBE2M and UBE2F N-terminal motifs, based on a conserved ^M[IL].L core sequences. These peptides bind into a deep conserved hydrophobic pocket of DCNLs PONY domain (Monda,2013, 4P5O, 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. Consequently these motifs are part of a switching mechanism. For the DNTL interaction, a N-terminal acetylation of the binding motif is required in the case of both E2s. The acetyl group neutralises a positive charge at the E2 N-terminus to ensure its penetration into the hydrophobic pocket within the PONY domain and additionally makes a positive interaction in the pocket. The methionine side chain of the E2 extends into a hydrophobic channel formed by DCNL side chains. In vitro studies show that all of the DCNLs are able to interact with both NEDD8 E2s, however with varying affinities. The highest affinities were observed for UBE2F~DCNL3, UBE2M~DCNL1 and UBE2M~DCNL2. DCNL1 amplification and overexpression in squamous cell carcinoma is associated with unfavourable outcome (Monda,2013). This raises the possibility, that the N-acetyl-methionine binding site may be an important drug target and shows the importance of understanding the binding mechanism for the design of small molecules to target the binding site. Due to differences in the N-acetyl-Met binding pockets between the UBE2F~DCNL3, UBE2M~DCNL1 and UBE2M~DCNL2 structures, and the differential preferences of peptides derived from UBE2M or UBE2F in inhibiting DCNL1-DCNL3-activated NEDD8 ligation, the potential of selective manipulation of N-acetyl-Met-dependent protein-protein interactions was shown (Monda,2013).