Role of an N-terminal site of Ubc9 in SUMO-1, -2, and -3 binding and conjugation.
Covalent posttranslational modification of target proteins with ubiquitin and ubiquitin-like proteins regulates many important cellular processes. However, the molecular mechanisms by which these proteins are activated and conjugated to substrates has yet to be fully understood. NMR studies have shown that the ubiquitin-like proteins SUMO-1, -2, and -3 interact with the same N-terminal region of the E2 conjugating enzyme Ubc9 with similar affinities. This is correlated to their almost identical utilization by Ubc9 in the SUMO conjugation pathway. To investigate the functional significance of this interaction, site-directed mutagenesis was used to alter residues in the SUMO binding surface of Ubc9, and the effect of the amino acid substitutions on binding and conjugation to SUMO-1 and target protein RanGAP1 was investigated by isothermal titration calorimetry and biochemical analysis. R13A/K14A and R17A/K18A mutations in Ubc9 disrupted the interaction with SUMO-1 but did not completely abolish the interaction with E1. While these Ubc9 mutants displayed a significantly reduced efficiency in the transfer of SUMO-1 from E1 to E2, their ability to recognize substrate and transfer SUMO-1 from E2 to the target protein was unaffected. These results suggest that the noncovalent binding site of SUMO-1 on Ubc9, although distant from the active site, is important for the transfer of SUMO-1 from the E1 to the E2. The conservation of E2 enzymes across the ubiquitin and ubiquitin-like protein pathways indicates that analogous N-terminal sites of E2 enzymes are likely to have similar roles in general.
Pubmed ID: 12924945 RIS Download
Amino Acid Sequence | Binding Sites | Binding, Competitive | GTPase-Activating Proteins | Glutathione Transferase | Humans | Ligases | Magnetic Resonance Spectroscopy | Models, Molecular | Molecular Sequence Data | Mutagenesis, Site-Directed | Mutation | Protein Binding | Protein Conformation | Recombinant Fusion Proteins | SUMO-1 Protein | Sequence Homology, Amino Acid | Small Ubiquitin-Related Modifier Proteins | Structure-Activity Relationship | Substrate Specificity | Ubiquitin | Ubiquitin-Conjugating Enzymes | Ubiquitins