60th residues of ubiquitin and Nedd8 are located out of E2-binding surfaces, but are important for K48 ubiquitin-linkage.
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.
Division of Magnetic Resonance, Korea Basic Science Institute Ochang Campus, Cheongwon-Gun, Ochang-Eup, Yangcheong-Ri 804-1, Chungcheongbuk-Do 363-883, South Korea.
Yun-Seok
YS
Choi
Young Ho
YH
Jeon
Kyoung-Seok
KS
Ryu
Chaejoon
C
Cheong
D000595:Amino Acid Sequence; D001665:Binding Sites; D008239:Lysine / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054875:Ubiquitination; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism
D014452:Ubiquitins; D008239:Lysine