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TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane
Anchoring Complexes of the Type VI Secretion System
Abdelrahim Zoued, Eric Durand, Cecilia Bebeacua, Yannick Brunet, Badreddine Douzi, Christian Cambillau, E. Cascales, Laure Journet
To cite this version:
Abdelrahim Zoued, Eric Durand, Cecilia Bebeacua, Yannick Brunet, Badreddine Douzi, et al..
TssK Is a Trimeric Cytoplasmic Protein Interacting with Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System. Journal of Biological Chem- istry, American Society for Biochemistry and Molecular Biology, 2013, 288 (38), pp.27031-27041.
�10.1074/jbc.M113.499772�. �hal-02091435�
Laure Journet
Douzi, Christian Cambillau, Eric Cascales and Bebeacua, Yannick R. Brunet, Badreddine Abdelrahim Zoued, Eric Durand, Cecilia
System Complexes of the Type VI Secretion
Phage-like and Membrane Anchoring Interacting with Components of Both TssK Is a Trimeric Cytoplasmic Protein
doi: 10.1074/jbc.M113.499772 originally published online August 6, 2013 2013, 288:27031-27041.
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TssK Is a Trimeric Cytoplasmic Protein Interacting with
Components of Both Phage-like and Membrane Anchoring Complexes of the Type VI Secretion System * □ S
Received for publication, July 5, 2013, and in revised form, August 5, 2013 Published, JBC Papers in Press, August 6, 2013, DOI 10.1074/jbc.M113.499772
Abdelrahim Zoued ‡1 , Eric Durand §2 , Cecilia Bebeacua §3 , Yannick R. Brunet ‡1 , Badreddine Douzi § , Christian Cambillau § , Eric Cascales ‡ , and Laure Journet ‡4
From the ‡ Laboratoire d’Ingénierie des Systèmes Macromoléculaires, Institut de Microbiologie de la Méditerranée, CNRS, UMR 7255, Aix-Marseille Université, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France and the § Architecture et
Fonction des Macromolécules Biologiques, Campus de Luminy, Case 932, Aix-Marseille Université, CNRS UMR 6098, 13288 Marseille Cedex 09, France
Background: The T6SS assembles from 13 proteins that form two sub-assemblies.
Results: TssK is a cytoplasmic protein that interacts with Hcp, TssC, TssL, and TssA.
Conclusion: The TssK complex is three-arm shaped and links the membrane and phage-like complexes in T6SS.
Significance: The structural and functional characterization of TssK leads to a better understanding of T6SS assembly.
The Type VI secretion system (T6SS) is a macromolecular machine that mediates bacteria-host or bacteria-bacteria inter- actions. The T6SS core apparatus assembles from 13 proteins that form two sub-assemblies: a phage-like complex and a trans- envelope complex. The Hcp, VgrG, TssE, and TssB/C subunits are structurally and functionally related to components of the tail of contractile bacteriophages. This phage-like structure is thought to be anchored to the membrane by a trans-envelope complex composed of the TssJ, TssL, and TssM proteins. How- ever, how the two sub-complexes are connected remains unknown. Here we identify TssK, a protein that establishes con- tacts with the two T6SS sub-complexes through direct interac- tions with TssL, Hcp, and TssC. TssK is a cytoplasmic protein assembling trimers that display a three-armed shape, as revealed by TEM and SAXS analyses. Fluorescence microscopy experi- ments further demonstrate the requirement of TssK for sheath assembly. Our results suggest a central role for TssK by linking both complexes during T6SS assembly.
Bacteria have developed macromolecular nanomachines to inject toxins to either prokaryotic or eukaryotic cells to clear an
environmental niche or to cause diseases (1). One of the sys- tems able to deliver toxic effectors to both prokaryotic and eukaryotic cells is the Type VI secretion system (T6SS) 5 (2). The current model regarding T6SS function proposes that it acts similarly to the cell puncturing device of contractile bacterio- phages (3–5).
Although several accessory proteins are usually necessary for its function, the T6SS assembles from 13 Tss (Type six sub- units) proteins whose genes are conserved in all T6SS gene clusters and are thus called “core components” (6 – 8). A num- ber of these conserved subunits share structural and functional similarities with contractile bacteriophage tail proteins includ- ing components of the tail tube, sheath, hub, and baseplate (4, 9 –12). For these reasons the structure assembled by these pro- teins and visualized by cryo-electron tomography and fluores- cence microscopy has been called the “phage-like complex.” A second group of core components are the TssJ, TssM, and TssL membrane proteins that assemble a trans-envelope spanning complex. Aside these two groups, the TssA, TssF, TssG, and TssK core components have not been characterized yet.
As briefly described above, the phage-like sub-complex is structurally and functionally similar to the contractile tail of Myoviridae. The crystal structures of the Hcp (hemolysin co- regulated protein) proteins present high similarity with the bac- teriophage tail tube gpV protein (10, 13–15). Six Hcp assem- ble into rings of about 80 –90 Å wide with an internal diameter of about 30 – 40 Å (13–15) that stack on each other to form tubes in vitro (9, 13–16). The VgrG (valine glycine repeat pro- tein) trimer is structurally homologous to the bacteriophage T4 gp27/gp5 spike complex which is used as a puncturing device to perforate the host cell envelope (9, 17). TssE is an homolog of gp25, a structural component of the phage baseplate (6, 18)
* This work was supported in part by the Centre National de la Recherche Scientifique (CNRS), the Aix-Marseille University, and a grant from the Agence Nationale de la Recherche (ANR-10-JCJC-1303-03) (to E. C.). This work was also supported by the CNRS, the Aix-Marseille University, and by grants from the Marseille-Nice Genopole, IBiSA, and the Fondation de la Recherche Médicale (FRM DEQ2011-0421282) (to C. C.).
□
SThis article contains supplemental Tables S1-S2 and Fig. S1.
1
Supported by doctoral fellowships from the French Ministère de la Recherche.
2
Supported by a post-doctoral fellowship from the Fondation pour la Recher- che Médicale (SPF20101221116).
3
Present address: Structural and Computational Biology & Cell Biology and Biophysics, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
4
To whom correspondence should be addressed: Laboratoire d’Ingénierie des Systèmes Macromoléculaires (UMR7255), CNRS, Aix-Marseille Univer- sité, 31 Chemin Joseph Aiguier, 13402 Marseille cedex 20, France. Tel.:
33491164156; Fax: 33491712124; E-mail: ljournet@imm.cnrs.fr.
5