Nedd8, a ubiquitin-like modifier, is covalently attached to various proteins. Although Nedd8 has higher sequence identity (57%) with ubiquitin, its conserved K48 residue cannot form covalent linkage with ubiquitin. To decipher the reason why Nedd8 cannot be an effective ubiquitin-acceptor, we compared the non-covalent interaction between Nedd8 and ubiquitin for various E2s using cross-saturation NMR technique. However, both Nedd8 and ubiquitin displayed almost identical non-covalent E2-binding properties. The K60 of Nedd8 was not present at the E2-binding surface, but its mutation to Asn converted Nedd8 into a ubiquitin-acceptor. The N60 ubiquitin mutants also displayed a decreased ubiquitin-accepting activity. These results suggest the presence of an uncharacterized determinant for the K48 ubiquitin-linkage that is not related to non-covalent E2-bindings.