PMID:18583342

Citation	Gébus, C, Caroline, G, Faudry, E, Eric, F, Bohn, YS, Elsen, S, Sylvie, E and Attree, I (2008) Oligomerization of PcrV and LcrV, protective antigens of Pseudomonas aeruginosa and Yersinia pestis. J. Biol. Chem. 283:23940-9
Abstract	Protective antigens of Pseudomonas aeruginosa (PcrV) and Yersinia pestis (LcrV) are key elements of specialized machinery, the type III secretion system (T3SS), which enables the injection of effector molecules into eukaryotic cells. Being positioned at the injectisome extremity, V proteins participate in the translocation process across the host cell plasma membrane. In this study, we demonstrate the assembly of V proteins into oligomeric doughnut-like complexes upon controlled refolding of the proteins in vitro. The oligomeric nature of refolded PcrV was revealed by size exclusion chromatography, native gel electrophoresis, and native mass spectrometry, which ascertain the capacity of the protein to multimerize into higher-order species. Furthermore, transmission electron microscopy performed on oligomers of both PcrV and LcrV revealed the presence of distinct structures with approximate internal and external diameters of 3-4 and 8-10 nm, respectively. The C-terminal helix, alpha12, of PcrV and notably the hydrophobic residues Val(255), Leu(262), and Leu(276) located within this helix, were shown to be crucial for oligomerization. Moreover, the corresponding mutant proteins produced in P. aeruginosa were found to be non-functional in in vivo type III-dependent cytotoxicity assays by directly affecting the correct assembly of PopB/D translocon within the host cell membranes. The detailed understanding of structure-function relationships of T3SS needle tip proteins will be of value in further developments of new vaccines and antimicrobials.
Links	PubMed PMC3259777 Online version:10.1074/jbc.M803146200
Keywords	Animals; Anti-Infective Agents/chemistry; Antigens, Bacterial/chemistry; Antigens, Bacterial/immunology; Bacterial Toxins/chemistry; Bacterial Toxins/immunology; Bacterial Vaccines/immunology; Drug Design; Hydrophobic and Hydrophilic Interactions; Macrophages/immunology; Mice; Multiprotein Complexes/chemistry; Multiprotein Complexes/immunology; Multiprotein Complexes/ultrastructure; Mutation; Plague/immunology; Pore Forming Cytotoxic Proteins/chemistry; Pore Forming Cytotoxic Proteins/immunology; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Pseudomonas Infections/immunology; Pseudomonas aeruginosa/chemistry; Pseudomonas aeruginosa/immunology; Structure-Activity Relationship; Yersinia pestis/chemistry; Yersinia pestis/immunology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
PSEAE:Q9I324		GO:0005576: extracellular region	ECO:0000314:		C		Figure 6 The presence of this protein in host cell was determined by immunodetection in sheep red blood cell membrane fractions.	complete

References

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