Convergent evolution in the assembly of polyubiquitin degradation signals by the Shigella flexneri IpaH9.8 ligase.

Autor:	Edwards DJ; From the Department of Biochemistry and Molecular Biology and., Streich FC Jr; From the Department of Biochemistry and Molecular Biology and., Ronchi VP; From the Department of Biochemistry and Molecular Biology and., Todaro DR; From the Department of Biochemistry and Molecular Biology and., Haas AL; From the Department of Biochemistry and Molecular Biology and the Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, New Orleans, Louisiana 70112 ahaas@lsuhsc.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2014 Dec 05; Vol. 289 (49), pp. 34114-28. Date of Electronic Publication: 2014 Oct 23.
DOI:	10.1074/jbc.M114.609164
Abstrakt:	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. (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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