Featured Article - January 2009
Short description: A structurally and mechanically distinct class of ubiquitin ligases has been discovered, and a single mutation changes this enzyme from a ligase to a potent thioesterase.Nature Struct. Mol. Biol. 15, 1293-1301 (2008)
Shigella flexneri is a food-borne bacterium that causes dysentery in humans. It does this by injecting effector proteins into the cells of its host by way of the bacterium's type III secretion system. One group of these effectors are the IpaH proteins, which affect the host cell's ubiquitination pathway, an emerging target of pathogenic bacteria.
Ubiquitination is involved in many different processes within the cell, most commonly as a tag that targets a protein for degradation in a proteasome. The addition of one or several ubiquitin molecules to a target protein requires a three-enzyme cascade. The C-terminal glycine residue of ubiquitin is first charged via a highly reactive thioester linkage to a cysteine residue in a ubiquitin-activating enzyme (E1). The E1-bound ubiquitin is then transferred to a cysteine residue on a ubiquitin-conjugating enzyme (E2). Finally, ubiquitin ligase (E3) brings the substrate and ubiquitin together, enabling the transfer of ubiquitin to a lysine residue on the substrate.
IpaH proteins have previously been shown to have ubiquitin ligase (E3) activity, but as the sequence of their C-terminal domain did not resemble that of any known E3s the nature and mechanism of this reaction was not clear.
It was thought that E3s fell into one of two classes. The first class have the RING (really interesting new gene) motif or a modified form of it, and they act as adaptors, bringing ubiquitin-charged E2 and the substrate close enough together to promote ubiquitination. The second class has a HECT (homologous to E6-associated protein C terminus) domain, which has an essential cysteine that acts as an acceptor for ubiquitin before its transfer to the substrate.
Two groups now report the structure of two IpaH proteins and provide biochemical insight into how these proteins work. Zhu et al. 1 solved the full-length structure of IpaH3 and Singer et al. 2 solved the C-terminal domain of IpaH1.4, whose sequence is almost identical to the C-terminal domains of all Shigella IpaH proteins. Both groups show that the C-terminal domain contains the catalytic activity for ubiquitin transfer and that this C-terminal domain has an all-helical fold with considerable flexibility that bears no resemblance to other E3 ubiquitin ligases. PSI MCSG contributed to the work of Singer et al.
Both groups demonstrated that Cys363 is essential for the ligase activity. Zhu et al. showed that it acts as a nucleophile to catalyze ubiquitin transfer through a transthiolation reaction and Singer et al. examined the effect in sst2Δ yeast of mutating this residue to an inactive cysteine. In sst2Δ yeast, the pheromone-response pathway is disrupted when the target of the ligase, Ste7, is ubiquitinated and degraded; this serves as a useful marker for IpaH's effects.
The results from both groups strongly suggest that IpaH enzymes use Cys363 as an acceptor of ubiquitin from E2s and then transfer the ubiquitin to a target protein.
In addition to the active-site cysteine, both groups identified other residues important for activity. Surprisingly, Zhu et al. found that replacing Asp365 with asparagine increased the speed of hydrolysis of ubiquitin charged with the E2 enzyme UbcH5c and detected increased amounts of free ubiquitin. This activity seems to require Cys363, as a double mutation of the cysteine and Asp365 was completely inactive. This is the first example of a single mutation turning an E3 ligase into a ubiquitin-E2 thioesterase.
These findings raise the question of whether there are other E3s waiting to be identified in prokaryotes.
Singer lexander U., Rohde John R., Lam Robert, Skarina Tatiana, Kagan Olga et al. Structure of the Shigella T3SS effector IpaH defines a new class of E3 ubiquitin ligases.
Nature Struct. Mol. Biol. 15, 1293-1301 (2008).
Zhu Yongqun, Li Hongtao, Hu Liyan, Wang Jiayi, Zhou Yan et al. Structure of a Shigella effector reveals a new class of ubiquitin ligases.
Nature Struct. Mol. Biol. 15, 1302-1308 (2008).