Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 ligase [Protein Synthesis and Degradation]

October 23rd, 2014 by Edwards, D. J., Streich, F. C., Ronchi, V. P., Todaro, D. R., Haas, A. L.

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 ten such effectors mimics ubiquitin ligases but bear no sequence or structural homology to their eukaryotic counterpoints. Using rates of 125I-polyubiquitin chain formation as a functional read out, IpaH9.8 displays V-type positive cooperativity with respect to varying concentrations of its Ubc5B~125I-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~125I-ubiquitin thioester (Ki =117±29 nM) while 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 (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 Phe395. 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 et al. (2013) J. Biol. Chem. 288, 10349-10360] suggest convergent evolution of the catalytic mechanisms for prokaryotic and eukaryotic ligases.
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