In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 27 ( 2012-07-03)
Abstract:
Finally, because Cif modulates ubiquitin-mediated protein degradation, detailed understanding of its activity paves the way for developing molecular tools to probe this important and highly conserved eukaryotic signaling pathway. Using these assays, we have identified the key features of the Cif/NEDD8 interaction, defining a surface at the “front line” of host-pathogen interactions. However, why would a pathogen take the trouble of making and delivering an inhibitor of the host cell cycle? This is a question that remains to be fully answered, but we speculate that Cif might slow the multiplication of gut epithelial cells (which could be important for food-borne pathogens, such as enteropathogenic Escherichia coli and Y. pseudotuberculosis ), delaying their renewal, and therefore favoring bacterial colonization. By inhibiting protein degradation pathways, Cif might also boost the activity of other effector proteins delivered to host cells at the same time. We then tested whether the complex formed in the protein crystals was relevant in solution by using mutagenesis techniques to change some of the amino acid residues of Cif that form the interface with NEDD8. Interestingly, we needed to make some dramatic changes to prevent complex formation. We concluded that complementarity in overall shape, rather than any specific individual interaction, is the driving force behind complex formation. We also used native gel electrophoresis to investigate the activity of Cif Yp and selected mutants of this protein. This, combined with the structure of the complex, has given us insights into the mechanism of deamidation. To this end, we determined the structures of complexes between the enzymes of this family and their substrate NEDD8 by using X-ray crystallography. We were successful in two cases, including Cif from Yersinia pseudotuberculosis (Cif Yp ), a mostly food-borne pathogen that adheres to intestinal cells before invasion and can cause pseudotuberculosis. These structures revealed a virtually identical, shared interaction surface. We also found that Cif family members bound NEDD8 in solution, and we hypothesize that all Cifs will bind NEDD8 similarly. The interface is composed of a variety of macromolecular interactions, including hydrogen bonds and hydrophobic contacts. The substrate glutamine residue is positioned in the active site of the enzyme in a conformation consistent with catalysis. The interaction between Cif and NEDD8 is not a simple “lock and key” but an “induced fit.” On complex formation, the C-terminal residues of NEDD8 are displaced from their position observed in the free (uncomplexed) molecule. This conformational change is enabled by the so-called “occluding-loop” in Cifs, an adaptation to the protein fold on which the structure of these enzymes is based. This allows the substrate glutamine to access the active site. To date, the activity of two members of the Cif family has been investigated in detail, and they both convert a specific amino acid residue on the target molecules from a glutamine to a glutamate [a deamidation reaction ( 4 )]. This correlates with dysfunction of ubiquitin chain formation and CRL activity, which is regulated by NEDD8, locking the CRLs into an inactive form (see Fig. P1 ). Here, we build on these exciting findings by revealing how Cifs recognize their substrates and provide insights into the catalytic mechanism. One host cell function that is targeted by pathogen effector proteins is the cell cycle. In eukaryotic cells, the cell cycle is split into discrete phases, and crossing the boundaries between these phases, known as checkpoints, requires specific conditions that are analogous to the requirement of a passport to cross national boundaries. When these checkpoints fail, uncontrolled cell division can occur, which can lead to cancer. One important mechanism in cell cycle regulation is the removal of individual proteins from the intracellular environment at specific times. For example, interfering with the system known as the ubiquitin-mediated protein degradation pathway can lead to an arrest of the cell cycle, halting cell division. In particular, dysfunction of the enzymes responsible for enabling this system, the most prominent of which are called cullin-RING ligases (CRLs), can lead to accumulation of factors whose removal is required for efficient cell cycle progression. Pathogenic bacteria use many different mechanisms to enhance their infectivity, one of which involves delivery of virulence factors, including effector proteins, into host cells via a molecular “syringe-and-needle” secretion system ( 1 ). The effector proteins modulate host cell functions ( 2 ) that presumably benefit the pathogen’s life cycle. The cycle inhibiting factor (Cif) family of bacterial effector proteins arrest the eukaryotic cell cycle ( 3 ) by targeting ubiquitin and the ubiquitin-like protein NEDD8 ( 4 , 5 ). To understand better how Cif enhances bacterial virulence, we determined and report here the X-ray crystal structures of Cif/NEDD8 complexes.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1112107109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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