Molecular Dissection of the Interface between the Type VI Secretion TssM Cytoplasmic Domain and the TssG Baseplate Component. Issue 22 (6th November 2016)

• Record Type: Journal Article
• Authors: Logger, Laureen; Aschtgen, Marie-Stéphanie; Guérin, Marie; Cascales, Eric; Durand, Eric
• Is Part Of: Journal of molecular biology. Volume 428:Issue 22(2016:Nov. 06)
• Page Start: 4424
• Page End: 4437
• Publication Date: 2016-11-06
• Subjects: T6SS type VI secretion system -- BP baseplate -- MC membrane complex -- IM inner membrane -- OM outer membrane -- EAEC enteroaggregative Escherichia coli -- TMH transmembrane helices -- NTPase nucleotide triphosphatase -- NTP nucleotide triphosphate -- DPY-30 Dumpy-30 -- MPB 3-(N-maleimidylpropionyl) biocytin -- WT wild-type -- SIM sci-1-inducing medium -- AHT anhydrotetracyclin -- PBS phosphate-buffered saline -- spGFP super folder Green Fluorescent Protein -- DMSO Diméthylsulfoxyde -- GTP Guanosine triphosphate; protein transport -- protein secretion -- type VI secretion -- bacterial competition -- membrane complex; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2016.08.032
• Languages: English
• ISSNs: 0022-2836

Abstract

Abstract: The type VI secretion system (T6SS) is a multiprotein complex that catalyses toxin secretion through the bacterial cell envelope of various Gram-negative bacteria including important human pathogens. This machine uses a bacteriophage-like contractile tail to puncture the prey cell and inject harmful toxins. The T6SS tail comprises an inner tube capped by the cell-puncturing spike and wrapped by the contractile sheath. This structure is built on an assembly platform, the baseplate, which is anchored to the bacterial cell envelope by the TssJLM membrane complex (MC). This MC serves as both a tail docking station and a channel for the passage of the inner tube. The TssM transmembrane protein is a key component of the MC as it connects the inner and outer membranes. In this study, we define the TssM topology, highlighting a large but poorly studied 35-kDa cytoplasmic domain, TssMCyto, located between two transmembrane segments. Protein–protein interaction assays further show that TssMCyto oligomerises and makes contacts with several baseplate components. Using computer predictions, we delineate two subdomains in TssMCyto, including a nucleotide triphosphatase (NTPase) domain, followed by a 110-aa extension. Finally, site-directed mutagenesis coupled to functional assays reveals the contribution of these subdomains and conserved motifs to the interaction with T6SS partners and to the function of the secretion apparatus. Graphical Abstract: Highlights: The T6SS TssM protein is constituted of three transmembrane segments. Transmembrane helices 2 and 3 delimitate a 35-kDa cytoplasmic domain. The TssM cytoplasmic loop is subdivided into two domains with NTPase and DPY30 folds. The TssM NTPase domain is lacking the catalytic site but interacts with TssK. The TssM DPY30 domain mediates oligomerisation and interaction with TssG.