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Identification of a substrate recognition site on Ubc9
Donghai Lin1, Michael H Tatham, Bin Yu
1Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
The Journal of Biological Chemistry
|March 6, 2002
Summary
Human Ubc9 conjugates small ubiquitin-like modifier 1 (SUMO-1), similar to ubiquitin pathways. Mutations reveal a Ubc9 substrate binding site, indicating target protein secondary structure isn't essential for SUMO-1 conjugation.
Area of Science:
- Biochemistry
- Molecular Biology
- Post-translational Modifications
Background:
- Human Ubc9 is a ubiquitin-conjugating enzyme homologous to E2 enzymes.
- It conjugates small ubiquitin-like modifier 1 (SUMO-1), not ubiquitin.
- The SUMO-1 conjugation pathway shares similarities with the ubiquitin pathway.
Purpose of the Study:
- To investigate the interaction between Ubc9 and SUMO-1 conjugation sites.
- To identify the substrate binding site on Ubc9.
- To determine the role of target protein secondary structure in SUMO-1 conjugation.
Main Methods:
- NMR spectroscopy to study Ubc9-substrate interactions.
- Site-directed mutagenesis of Ubc9 residues.
- Analysis of SUMO-1 conjugation to target proteins (p53, E1B, PML).
Main Results:
- SUMO-1 conjugation site peptides from p53 and c-Jun bind near Ubc9's active site Cys93.
- Mutations in this Ubc9 region significantly affect SUMO-1 conjugation.
- The SUMO-1 conjugation site of p53 lacks defined secondary structure when free or bound to Ubc9.
Conclusions:
- A specific substrate binding site on Ubc9 has been defined through mutagenesis.
- Defined secondary structure at target protein SUMO-1 conjugation sites is not required for recognition and conjugation.