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J. Biol. Chem., Vol. 283, Issue 35, 23940-23949, August 29, 2008

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Oligomerization of PcrV and LcrV, Protective Antigens of Pseudomonas aeruginosa and Yersinia pestis^*

From the Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (Unité mixte de recherche 5092), CNRS, Université Joseph Fourier, Commissariat à l'Energie Atomique (CEA), DSV, iRTSV, Grenoble, France

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, 12, of PcrV and notably the hydrophobic residues Val²⁵⁵, Leu²⁶², and Leu²⁷⁶ 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.

Received for publication, April 24, 2008 , and in revised form, June 4, 2008.

^* This work was supported in part by Research Contract 06.70.151. 00.470.75.96 from Délégation Générale pour l'Armement (DGA) and the French Cystic Fibrosis Association Vaincre la Mucoviscidose. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2.

¹ Supported by a Ph.D. fellowship from the Délégation Générale pour l'Armement.

² Supported by a postdoctoral fellowship from the Direction des sciences du vivant, Commissariat à l'Energie Atomique.

³ To whom correspondence should be addressed: iRTSV/BBSI, Commissariat à l'Energie Atomique Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 09, France. Tel.: 33-438783483; Fax: 33-438784499; E-mail: iattreedelic{at}cea.fr.

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