title: 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 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology and.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Edwards
authors.affiliation: From the Department of Biochemistry and Molecular Biology and.
authors.forename: Frederick C
authors.initials: FC
authors.lastname: Streich
authors.affiliation: From the Department of Biochemistry and Molecular Biology and.
authors.forename: Virginia P
authors.initials: VP
authors.lastname: Ronchi
authors.affiliation: From the Department of Biochemistry and Molecular Biology and.
authors.forename: Dustin R
authors.initials: DR
authors.lastname: Todaro
authors.affiliation: 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.
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Haas
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001667:Binding, Competitive; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D007457:Iodine Radioisotopes; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012763:Shigella flexneri / Q000737:chemistry* / Q000201:enzymology; D015398:Signal Transduction; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007457:Iodine Radioisotopes; D021122:Protein Subunits; D011994:Recombinant Proteins; C063500:ipaH protein, Shigella flexneri; D025821:Polyubiquitin; D044767:Ubiquitin-Protein Ligases
keywords: Conjugation; Cooperativity; E3 Ubiquitin Ligase; Enzyme Mechanism; IpaH; Oligomer; Polyubiquitin Chain; Protein Degradation; Ubc5; Virulence Factor