PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Protein Folding and Misfolding: It's the Journey, Not the Destination
March 2015
CCR5 and HIV Infection
January 2015
HIV/AIDS: Pre-fusion Env Exposed
January 2015
HIV/AIDS: Slide to Enter
January 2015
Updating ModBase
January 2015
Power in Numbers
August 2014
Quorum Sensing: A Groovy New Component
August 2014
Bacterial CDI Toxins
June 2014
Immunity: One Antibody to Rule Them All
June 2014
Virology: A Bat Influenza Hemagglutinin
March 2014
Virology: Making Sensitive Magic
March 2014
Virology: Visualizing Cyanophage Assembly
March 2014
Virology: Zeroing in on HBV Egress
March 2014
Viroporins
March 2014
Cas4 Nuclease and Bacterial Immunity
February 2014
Microbial Pathogenesis: A GNAT from Pseudomonas
February 2014
Microbial Pathogenesis: Targeting Drug Resistance in Mycobacterium tuberculosis
February 2014
Microbiome: The Dynamics of Infection
September 2013
Membrane Proteome: A Funnel-like Viroporin
August 2013
Infectious Diseases: A Pathogen Ubiquitin Ligase
May 2013
Infectious Diseases: A Shared Syringe
May 2013
Infectious Diseases: Determining the Essential Structome
May 2013
Infectious Diseases: Targeting Meningitis
May 2013
NDM-1 and Antibiotics
May 2013
Bacterial Hemophores
January 2013
Microbial Pathogenesis: Computational Epitope Prediction
January 2013
Microbial Pathogenesis: Influenza Inhibitor Screen
January 2013
Microbial Pathogenesis: Measles Virus Attachment
January 2013
Microbial Pathogenesis: NEAT Iron
January 2013
Membrane Proteome: Sphingolipid Synthesis Selectivity
December 2012
A signal sensing switch
September 2012
Gauging needle structure
July 2012
Anthrax Stealth Siderophores
June 2012
A Pseudomonas L-serine dehydrogenase
May 2012
Pilus Assembly Protein TadZ
April 2012
Making Lipopolysaccharide
January 2012
Superbugs and Antibiotic Resistance
December 2011
A change to resistance
November 2011
An effective and cooperative dimer
November 2011
The Perils of Protein Secretion
November 2011
Bacterial Armor
October 2011
Breaking down the defenses
September 2011
Moving some metal
August 2011
Capsid assembly in motion
April 2011
Know thy enemy … structurally
October 2010
Treating sleeping sickness
May 2010
Bacterial spore kinase
April 2010
Hemolysin BL
January 2010
Unusual cell division
October 2009
Anthrax evasion tactics
September 2009
Toxin-antitoxin VapBC-5
September 2009
Antibiotic target
August 2009
Lysostaphin
July 2009
Tackling influenza
June 2009
You look familiar: the Type VI secretion system
June 2009
Unique SARS
April 2009
Anthrax stealth molecule
March 2009
A new class of bacterial E3 ubiquitination enzymes
January 2009
Antiviral evasion
October 2008
SARS connections
September 2008
SARS Coronavirus Nonstructural Protein 1
June 2008

Research Themes Infectious diseases

A new class of bacterial E3 ubiquitination enzymes

PSI-SGKB [doi:10.1038/fa_psisgkb.2008.21]
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)

Surface representation of the C-terminal domain of IpaH1.4 with the partially transparent surface enclosing a ribbon representation of the molecule. Image courtesy of Ontario Centre for Structural Proteomics. (IpaH1.4 PDB 3CKD; IpaH3 PDB 3CVR

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.

Maria Hodges

References

  1. 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).

  2. 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).

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health