Community Research and Development Information Service - CORDIS



Project ID: 615984
Funded under: FP7-IDEAS-ERC
Country: Germany

Mid-Term Report Summary - BACTERIAL SYRINGES (Protein Translocation Through Bacterial Syringes)

The BACTERIAL SYRINGES project aims at understanding the molecular basis of cellular infection by bacterial ABC-type Tc toxins. In our initial cryo-EM work on the P. luminescens Tc complex we discovered that Tcs use a special syringe-like device for cell entry. Building on these results, we unraveled the molecular mechanism through which this unusual and complicated injection system allows membrane permeation and protein translocation. We extended our research to X-ray crystallography complemented by cryo-EM to obtain structures of the 1.7 MDa toxin at atomic resolution. We obtained well-diffracting crystals of the TcA as well as of the TcB and TcC subunits and solved the structures by molecular replacement using our cryo-EM structure as model.
The structures revealed that TcB and TcC (TcdB2 and TccC3) form a cocoon, in which the C-terminal region of TcC is autoproteolytically cleaved. Pentameric TcA forms a translocation channel, which is surrounded by a shell that comprises four putative receptor-binding sites and a neuraminidase-like region, probably important for the host specificity of the respective toxins. In addition, we showed that a pH-induced opening of the shell releases a putative entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed ß-propeller and results in a continuous protein translocation channel. This mechanism is unique and specific for Tc toxins and differs from that of other PFTs.
In our most recent work, we have embedded TcA in nanodiscs to ensure a natural lipid environment and stability of TcA. We then used cryo-EM with direct electron detection and single particle analysis to determine the structure of TcA in its pore state. The 3.5 Å structure reveals that the transmembrane helices rearrange in the membrane and open the initially closed pore, similarly to an iris diaphragm. However, they do not span the membrane completely. The edge of the channel is formed by extended loops instead that interact with lipid head groups and reach just to the surface of the membrane. To our knowledge, it is the first time that the transmembrane region of a Tc toxin and in general of a PFT is described in its near-to-native environment in atomic detail.

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