ARTICLE https://doi.org/10.1038/s41586-018-0556-6 Tc toxin activation requires unfolding and refolding of a β-propeller Christos Gatsogiannis 1,3 , Felipe Merino 1,3 , Daniel Roderer 1,3 , David Balchin 2 , Evelyn Schubert 1 , Anne Kuhlee 1 , Manajit Hayer-Hartl 2 & Stefan Raunser 1 * Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB–TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel. Tc toxins are found in pathogenic bacteria that affect insects and humans 1 . Tc toxins of insect pathogens are potential biopesticides and therefore the focus of crop protection research 2,3 , and understanding the mechanism of action of Tc toxins of human pathogens is medically relevant 4,5 . A Photorhabdus toxin complex (Tc) is typically composed of three proteins, TcA, TcB and TcC (Fig. 1a). TcB and TcC form a heterodimeric cocoon of about 300 kDa. The C-terminal hypervaria- ble region (HVR) of TcC 1 is autoproteolytically cleaved, generating a toxic enzyme of about 30 kDa 6,7 . The HVR varies greatly in sequence among different TcC homologues; the enzymes therefore have diverse toxic activities. In the case of TccC3, a TcC protein from Photorhabdus luminescens, the enzyme functions as an ADP-ribosyltransferase, which post-translationally modifies actin, leading to intracellular actin aggre- gation and cell death 8 . The enzyme is not resolved in the crystal struc- tures of either TccC3 or its Yersinia entomophaga homologue YenC2 6,7 , suggesting that it is at least partially unfolded inside the cocoon. TcA is a 1.4-MDa protein, which forms a translocation channel that is shielded by a shell 6,9 . A shift to higher or lower pH opens an electrostatic lock at the bottom of the shell, triggering its structural rearrangement 6,9,10 . The translocation channel is released and the compaction of a stretched linker that connects channel and shell drives the membrane insertion of TcA 6,10 . The anchoring of TcA on the membrane and the necessary counterforce for insertion are likely to be provided by binding to one or more receptors, which remain unidentified. Once inside the membrane, conformational changes result in the opening of the channel 10 . Structural studies of the wild-type ABC holotoxin (ABC(WT)) from the P. luminescens strain W14, comprising TcA (TcdA1), TcB (TcdB2) and TcC (TccC3), have demonstrated that binding of TcB–TcC to TcA induces the opening of a gate formed by a distorted six-bladed β-propeller at the bottom of TcB–TcC 6 . However, the mechanism of gate control and opening remain unknown. We previously hypoth- esized that after gate opening the ADP-ribosyltransferase inside the TcB–TcC cocoon is translocated into the TcA channel, and ultimately released into the host cell 6 . However, clear densities corresponding to the β-propeller and ADP-ribosyltransferase were missing in the elec- tron cryo-microscopy (cryo-EM) map of the holotoxin, limiting our understanding of the opening and initial translocation event. Here we present two near-atomic cryo-EM structures of a complete Tc holotoxin complex, which reveal the precise mechanism of Tc toxin assembly, gate opening and release of the cytotoxic enzyme into the translocation channel. 1 Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Dortmund, Germany. 2 Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany. 3 These authors contributed equally: Christos Gatsogiannis, Felipe Merino, Daniel Roderer. *e-mail: stefan.raunser@mpi-dortmund.mpg.de 370 Å TcA TcB TcC a TcB–TcC Holotoxin (TcA–TcB–TcC) Gate closed Gate open 90˚ b c Gatekeeper hairpin Hinge hairpin Sensor loop Sensor loop Blade 1 Blade 2 Blade 3 Blade 4 Blade 5 Blade 6 TcA- binding domain TcB- binding domain Fig. 1 | Cryo-EM structure of the ABC holotoxin. a, Side view of the 3D reconstruction of the ABC holotoxin complex (TcA (coloured by subunits), TcB (blue) and TcC (purple)). b, c, Side and top views of the closed (b, RCSB Protein Data Bank code (PDB) 4O9X 6 ) and open (c) state of the β-propeller domain of TcB. Blades 1, 2, 5 and 6 (salmon), blades 3 and 4 (gatekeeper domain, blue and purple), gatekeeper hairpin residues (residues 514–524, red), the sensor loop (residues 527–536, orange) and the TcB-binding domain of TcA (green) are highlighted. The TcB–TcC cocoon is coloured according to a. The β-hairpin 537–546 (hinge hairpin), next to the TcB sensor loop, opens by 90°. N AT U R E | www.nature.com/nature © 2018 Springer Nature Limited. All rights reserved.