Structure of the Vibrio cholerae Type VI secretion tubule at sub-nanometer resolution

The bacterial type VI secretion system is a multicomponent molecular machine directed against eukaryotic host cells and competing bacteria. It consists of a contractile tubule that is attached to a membrane protein complex. Upon tubule contraction, a needle is ejected into target cells to translocate toxic effectors into the cell. Due to structural and functional homologies of several proteins of the secretion system to proteins of contractile bacteriophage tails, the system is generally described as an inverted phage tail. Following this analogy, the secretion process is driven by energy stored in the elongated conformation of the Type VI secretion tubule for which also partial structural homology to bacteriophage tail sheath proteins has been predicted. However, this prediction has not been corroborated by structural data so far. The AAA+ ATPase ClpV plays an important role in the secretion process, as it disassembles the contracted tubule, putatively for recycling of the complex. Even though the binding site for ClpV has been identified in VipB, the molecular mechanism which recruits the ATPase specifically to the contracted tubule is not known yet. In a collaborative project with PD Dr. Axel Mogk and colleagues at the DKFZ Heidelberg and the group of Dr. Franz Herzog at the Gene Center Munich, we investigate the structure of the Vibrio cholerae Type VI secretion tubule consisting of the proteins VipA and VipB. We employ a hybrid methods approach of cryo electron microscopic 3D reconstruction and electron microscopic and biochemical labeling techniques supported by cross-linking mass spectrometry to develop a structural model of VipA and VipB in the tubule. We are able to resolve the three-dimensional structure of the helical VipA/B tubule up to 6 A which allows us to locate secondary structure elements. We describe the arrangement of VipA and VipB in the asymmetric unit and show that the architecture of the tubule is mainly defined by contacts between C-terminal domains of VipB which are structurally similar to domain IV of viral tail sheath proteins. By comparison to the T4 bacteriophage tail sheath, we suggest that these structurally homologous parts mediate the common function of contraction. Additionally, the VipA/B tubule has been adapted towards efficient recycling of contracted Type VI secretion systems. VipB is equipped with a specific four-helix bundle N-terminal domain which carries the ClpV binding motif. Also for VipA, no correspondency to any other known structural part of a phage-like contractile system is found. We propose that it serves as a chaperone for VipB. Based on the observed structural homologies between the T4 phage tail sheath protein and VipB, we model the elongated state of the VipA/B tubule using known low resolution structures of the elongated T4 phage tail. Furthermore, we suggest a molecular mechanism for Type VI secretion tubule recycling. In the elongated state of the tubule, the VipB N-terminal domain is hidden in the tubule wall, making the ClpV binding motif inaccessible for the ATPase. Therefore, ClpV-mediated recycling of the tubule is restricted to its contracted state.