Convergent Evolution in the Assembly of Polyubiquitin Degradation Signals by the Shigella flexneri IpaH9.8 ligase

Abstract 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.