Identification of a Multifunctional Binding Site on Ubc9p Required for Smt3p Conjugation
| Autor: | Clive A. Slaughter, Vishwajeeth Pagala, Brenda A. Schulman, Kalman P. Bencsath, Michael S. Podgorski |
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| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Magnetic Resonance Spectroscopy Saccharomyces cerevisiae Proteins Time Factors Stereochemistry DNA Mutational Analysis Saccharomyces cerevisiae Biology Conjugated system medicine.disease_cause Biochemistry Mass Spectrometry Ligases Ubiquitin medicine Sulfhydryl Compounds Cloning Molecular Binding site Molecular Biology chemistry.chemical_classification Mutation Binding Sites Lysine C-terminus Esters Cell Biology biology.organism_classification Protein Structure Tertiary Repressor Proteins Enzyme chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Ubiquitin-Conjugating Enzymes Small Ubiquitin-Related Modifier Proteins biology.protein Electrophoresis Polyacrylamide Gel Chromatography Liquid Protein Binding Conjugate |
| Zdroj: | Journal of Biological Chemistry. 277:47938-47945 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m207442200 |
| Popis: | Ubiquitin-like proteins (ub-lps) are conjugated by a conserved enzymatic pathway, involving ATP-dependent activation at the C terminus by an activating enzyme (E1) and formation of a thiolester intermediate with a conjugating enzyme (E2) prior to ligation to the target. Ubc9, the E2 for SUMO, synthesizes polymeric chains in the presence of its E1 and MgATP. To better understand conjugation of ub-lps, we have performed mutational analysis of Saccharomyces cerevisiae Ubc9p, which conjugates the SUMO family member Smt3p. We have identified Ubc9p surfaces involved in thiolester bond and Smt3p-Smt3p chain formation. The residues involved in thiolester bond formation map to a surface we show is the E1 binding site, and E2s for other ub-lps are likely to bind to their E1s at a homologous site. We also find that this same surface binds Smt3p. A mutation that impairs binding to E1 but not Smt3p impairs thiolester bond formation, suggesting that it is the E1 interaction at this site that is crucial. Interestingly, other E2s and their relatives also use this same surface for binding to ubiquitin, E3s, and other proteins, revealing this to be a multipurpose binding site and suggesting that the entire E1-E2-E3 pathway has coevolved for a given ub-lp. |
| Databáze: | OpenAIRE |
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