Identification of a substrate recognition site on Ubc9.
Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (E1), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys93 of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.
Lin
Donghai
D
Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Tatham
Michael H
MH
Yu
Bin
B
Kim
Suhkmann
S
Hay
Ronald T
RT
Chen
Yuan
Y
D001665:Binding Sites; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000737:chemistry*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D018076:DNA, Complementary; D010455:Peptides; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; D005982:Glutathione Transferase; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D003545:Cysteine