Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 Ligase
| Autor: | Frederick C. Streich, Arthur L. Haas, Dustin R. Todaro, Daniel J. Edwards, Virginia P. Ronchi |
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| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Stereochemistry Ubiquitin-Protein Ligases Gene Expression Cooperativity Substrate analog Protein degradation Biology Binding Competitive Biochemistry Shigella flexneri Substrate Specificity Evolution Molecular Iodine Radioisotopes chemistry.chemical_compound Allosteric Regulation Bacterial Proteins Escherichia coli Polyubiquitin Molecular Biology chemistry.chemical_classification Antigens Bacterial DNA ligase Cooperative binding Cell Biology biology.organism_classification Recombinant Proteins Ubiquitin ligase Kinetics Protein Subunits chemistry Mutation Enzymology biology.protein Protein Multimerization Allosteric Site Protein Binding Signal Transduction |
| Zdroj: | Journal of Biological Chemistry. 289:34114-34128 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m114.609164 |
| Popis: | The human pathogen Shigella flexneri subverts host function and defenses by deploying a cohort of effector proteins via a type III secretion system. The IpaH family of 10 such effectors mimics ubiquitin ligases but bears no sequence or structural homology to their eukaryotic counterpoints. Using rates of (125)I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B∼(125)I-ubiquitin thioester co-substrate in the nanomolar range ([S]½ = 140 ± 32 nm; n = 1.8 ± 0.1) and cooperative substrate inhibition at micromolar concentrations ([S]½ = 740 ± 240 nm; n = 1.7 ± 0.2), requiring ordered binding to two functionally distinct sites per subunit. The isosteric substrate analog Ubc5BC85S-ubiquitin oxyester acts as a competitive inhibitor of wild-type Ubc5B∼(125)I-ubiquitin thioester (Ki = 117 ± 29 nm), whereas a Ubc5BC85A product analog shows noncompetitive inhibition (Ki = 2.2 ± 0.5 μm), consistent with the two-site model. Re-evaluation of a related IpaH3 crystal structure (PDB entry 3CVR) identifies a symmetric dimer consistent with the observed cooperativity. Genetic disruption of the predicted IpaH9.8 dimer interface reduces the solution molecular weight and significantly ablates the kcat but not [S]½ for polyubiquitin chain formation. Other studies demonstrate that cooperativity requires the N-terminal leucine-rich repeat-targeting domain and is transduced through Phe(395). Additionally, these mechanistic features are conserved in a distantly related SspH2 Salmonella enterica ligase. Kinetic parallels between IpaH9.8 and the recently revised mechanism for E6AP/UBE3A (Ronchi, V. P., Klein, J. M., and Haas, A. L. (2013) E6AP/UBE3A ubiquitin ligase harbors two E2∼ubiquitin binding sites. J. Biol. Chem. 288, 10349-10360) suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases. |
| Databáze: | OpenAIRE |
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