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

Abstract

Background: IpaH bacterial ubiquitin ligases show no homology with eukaryotic ligases, and their mechanism is speculative. Results: IpaH9.8 functions as a cooperative allosteric dimer with two Ubc5ϳubiquitin binding sites per subunit. Conclusion: Kinetic parallels between IpaH and eukaryotic HECT ligases suggest convergent catalytic cycle evolution. Significance: These are the first mechanistic details of the IpaH enzyme catalytic mechanism.

The ubiquitin ligase SspH1 from Salmonella uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation

“…This suggests that at least a portion of helix 7 interacts with the CSD and these contacts aid in the stability and solubility of this subdomain. Purified NSD (residues 403-516) and CSD (residues 489 -700) constructs yield dispersed 1 H, 15 N TROSY NMR spectra (Fig. 2, A and B).…”

“…A previous study suggested that SspH/IpaH constructs containing both LRR and E3 domains form dimers in solution (15). Such high-molecular weight complexes could negatively impact NMR-based experiments and complicate data analysis.…”

“…Despite structural differences, SspH/IpaH E3s share important functional similarities with their eukaryotic HECT counterparts. Like HECT E3s, SspH/IpaH E3s form an E3ϳUb intermediate, use specific host E2ϳUbs, and target specific host proteins (12)(13)(14)(15)(16).…”

“…Although the structure of the SspH1 E3 domain has not yet been solved, those of several E3s, including the closely related SspH2 from Salmonella (77.6% identical in the E3 domain), are available. In addition, several studies have shown that SspH/IpaH E3s use E2ϳUbs from the Ube2D (UbcH5) family as a source of activated Ub (12)(13)(14)(15)(16)20). Thus, SspH1 is a structurally tractable system for investigating protein interactions and domain motions that occur during substrate ubiquitylation.…”

Midori‐ishi Cyan/monomeric Kusabira‐Orange‐based fluorescence resonance energy transfer assay for characterization of various E3 ligases

“…S1B in Supporting Information). k cat represents the number of times each enzyme site converts the substrate to the end product per unit time, whereas K m represents the E2 concentration needed to achieve a half-maximum elevation velocity for medi- ating ubiquitination (Edwards et al 2014). UBCH5A exhibited the highest IpaH4.5 activity relative to the other E2s ( Fig.…”