When NAE1:UBA3 is doubly loaded with NEDD8 (one molecule covalently attached to the catalytic cysteine and the other bound in the adenylation site), the E1 enzyme is competent to interact with either of its E2 enzymes, UBE2F and UBE2M (also known as UBC12). Three binding interfaces contribute to the interaction of the E1 and E2 enzymes. When doubly neddylated, the ubiquitin fold domain of NAE1 reorients and, in conjunction with the adenylation domain, forms a cryptic E2-binding site. The adenylation domain also makes contact with the amino terminus of either E2 enzyme. UBE2M additionally interacts directly with the NEDD8 molecule covalently attached to the E1 catalytic cysteine (Huang et al, 2004; Huang et al, 2007; reviewed in Enchev et al, 2015).
UBE2F is the E2 responsible for the transfer of NEDD8 to the CUL5:RBX2 E3 ligase complex, while UBE2M is specific for RBX1-containing E3 ligase complexes formed with CUL1-4 (Huang et al, 2009; reviewed in Mahon et al, 2014).