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.

Keywords: Conjugation; Cooperativity; E3 Ubiquitin Ligase; Enzyme Mechanism; IpaH; Oligomer; Polyubiquitin Chain; Protein Degradation; Ubc5; Virulence Factor.

FIGURE 1.

The Ubc5 family of E2 enzymes supports IpaH9.8-catalyzed assembly of unanchored lysine 48-linked polyubiquitin chains. A, ¹²⁵I-ubiquitin conjugation assays containing 5 nm IpaH9.8 were incubated for 10 min in the absence (lane 1) or presence (lanes 2–10) of a 100 nm concentration of the indicated E2 paralog and then quenched with sample buffer and resolved by 12% (w/v) SDS-PAGE under reducing conditions as described under “Materials and Methods.” The resulting gel was dried and visualized by autoradiography. B, an incubation identical to that of lane 4 of A was quenched by the addition of 8 IU of apyrase, and DTT was added to a final concentration of 10 mm to reduce all thioester bonds (0 min). Isopeptidase T was added to a final concentration of 3.8 μm by total protein, and aliquots were removed at the indicated times and quenched with sample buffer. At 50 min, additional isopeptidase T was added to a final concentration of 7.6 μm by total protein, and the reaction was allowed to continue for an additional 20 min (lane 8). The samples were resolved and visualized as in A. C, after autoradiography of the gel in B, ¹²⁵I-ubiquitin conjugates larger than 25 kDa were excised and quantified by γ-counting. The percentage of ¹²⁵I radioactivity remaining compared with zero time was analyzed by a semilog plot. The dashed line represents extrapolation of the limiting linear rate to t₀ in order graphically to determine the fraction of IsoT refractory (anchored) versus IsoT sensitive (unanchored) chains. D, ¹²⁵I-ubiquitin conjugation assays containing 5 nm IpaH9.8 were incubated for 10 min with 5 μm ¹²⁵I-ubiquitin (lane 3) or 1 μm ¹²⁵I-ubiquitin and a 4 μm concentration of the indicated unlabeled wild type or single lysine-to-arginine ubiquitin point mutant (lanes 4–11). Samples were resolved and visualized as in A. Mobility of relative molecular weight markers and position of the stacker gel are shown to the left. Position of free ¹²⁵I-ubiquitin is shown to the right.

FIGURE 2.

IpaH9.8 concentration dependence for ubiquitin chain partitioning. A, autoradiogram of ¹²⁵I-ubiquitin conjugation assays containing 15 nm Uba1 and 132 nm Ubc5A in the absence (lane 1) or presence (lanes 2–12) of varying amounts of IpaH9.8 (9.1 μm to 0.95 nm) were incubated for 10 min as described under “Materials and Methods.” Samples were resolved by 12% (w/v) SDS-PAGE and visualized by autoradiography. The mobility of relative molecular weight markers and position of the stacker gel are shown to the left. The position of free ¹²⁵I-ubiquitin is shown to the right. B, ¹²⁵I-ubiquitin conjugation reactions containing either 5 μm (closed circles) or 5 nm (open circles) IpaH9.8 were assayed for IsoT sensitivity as described in the legend to Fig. 1, B and C. The limiting rates for signal loss (dashed lines) were extrapolated to t₀ in order graphically to determine the fraction of IsoT labile conjugates as the y intercept.

FIGURE 3.

IpaH9.8 exhibits ordered cooperative allosteric kinetics. A, autoradiogram of 10-min ¹²⁵I-ubiquitin conjugation assays containing 2.5 nm IpaH9.8 in the absence (lane 1) or presence (lanes 2–9) of increasing concentrations of Ubc5B (5–500 nm) as described under “Materials and Methods.” The assay of lane 10 contained the highest concentration of Ubc5B and twice the concentration of Uba1 to verify IpaH9.8-limiting conditions. The assay of lane 11 was identical to that of lane 9 but in the absence of IpaH9.8. Gels were visualized by autoradiography and quantitated by γ-counting as described under “Materials and Methods.” B, concentration dependence of initial velocity versus [E2]_o for Ubc5A (closed circles), Ubc5B (open circles), and Ubc5C (closed squares). Lines, theoretical nonlinear sigmoidal regression fits of the data for kinetic constants summarized in Table 1. C, concentration dependence of initial velocity on [Ubc5B]_o. Line, nonlinear regression fit of the data to Equation 1 and the kinetic constants summarized under “Results,” where K′ = [S]_0.5^nH.

FIGURE 4.

IpaH9.8(244–545) exhibits hyperbolic kinetics. Shown is the concentration dependence of initial velocity versus [E2]_o for Ubc5A (closed circles), Ubc5B (open circles), and Ubc5C (closed squares) in the presence of 25 pm IpaH9.8(244–545) in assays otherwise identical to those of Fig. 3. Lines, theoretical nonlinear hyperbolic regression fits of the data for kinetic constants summarized in Table 2. Inset, double reciprocal plot of the data of Ubc5B.

FIGURE 5.

IpaH9.8 possesses two distinct Ubc5B∼ubiquitin thioester binding sites. A, double reciprocal plots of initial rates of ¹²⁵I-ubiquitin conjugation determined as in Fig. 3 under IpaH9.8-limiting conditions in assays containing 5 nm IpaH9.8 and the indicated concentrations of Ubc5B in the absence (closed circles) or presence (open circles) of 270 nm Ubc5BC85S-¹²⁵I-ubiquitin oxyester. B, double reciprocal plot of initial rates of ¹²⁵I-ubiquitin conjugation determined as in Fig. 3 under IpaH9.8-limiting conditions in assays containing 2.5 nm IpaH9.8 and the indicated concentrations of Ubc5B in the absence (closed circles) or presence (open circles) of 3.5 μm Ubc5BC85A product analog.

FIGURE 6.

Identification of a symmetric dimer in the crystal structure of the related IpaH3 ligase. A, surface rendering of the crystal structure for IpaH3 (PDB code 3CVR) in complex with its dimer mate identified by PISA analysis. Active site cysteine residues are colored yellow. B, ribbon diagram of the crystal structure for IpaH3 in complex with its dimer mate in the same orientation as in A. Residue side chains predicted by PISA analysis to be at the dimer interface are modeled as spheres. Active site cysteine residues are modeled as yellow spheres. C, gel filtration chromatography of IpaH9.8(244–545), as described under “Materials and Methods.” Inset, relative mobility of peaks 1 and 2 (closed circles) are shown on the calibration plot summarizing a series of standards (open circles). The predicted elution volume for monomeric IpaH9.8(244–545) is indicated (inset).

FIGURE 7.

IpaH9.8 dimer formation is necessary for activity. A, initial rates of ¹²⁵I-ubiquitin conjugation were determined as in Fig. 3 under E3-limiting conditions in assays containing the indicated concentrations of Ubc5B and either 2.5 nm IpaH9.8 (closed circles) or 5 nm IpaH9.8L435R (open circles). B, static light scattering analysis of 75 μm of either IpaH9.8 (solid line) or IpaH9.8L435R (dashed line). C, SDS-PAGE resolution and Coomassie Brilliant Blue staining of glutathione-Sepharose precipitation assays of reactions containing an 8.5 μm concentration of either IpaH9.8 (left) or IpaH9.8L435R (right) in the absence or presence of 8.5 μm either GST-IpaH9.8 or GST-IpaH9.8L435R, respectively. D, quantitation of the amount of IpaH9.8 or IpaH9.8L435R co-purifying with the respective GST-IpaH9.8 or GST-IpaH9.8L435R compared with background binding in the absence of the GST-fused proteins at the indicated times as analyzed by quantitative densitometry described under “Materials and Methods.”

FIGURE 8.

Cooperativity is transduced through Phe³⁹³. Initial rates of ¹²⁵I-polyubiquitin chain formation were determined as in Fig. 3 in the presence of 5 nm IpaH9.8F395A. Kinetic constants were determined by nonlinear regression analysis fit to hyperbolic kinetics. Inset, double reciprocal plot of the data.

FIGURE 9.

SspH2 exhibits cooperative kinetics. Concentration dependence of initial velocity versus [Ubc5B]_o in the presence of 5 nm SspH2. Line, theoretical sigmoidal nonlinear regression fit of the data for kinetic constants under “Results.” Inset, Hill plot for the data.