Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Cryo-EM structures of YenTc: changing paradigms of host interaction in pore-forming ABC toxins (#201)

Sarah Piper 1 , Lou Brillault 1 , Joseph Box 1 , Tristan Croll 2 , Jason Busby 3 , Shaun Lott 3 , Mark Hurst 4 , Glenn King 1 , Michael Landsberg 1
  1. University of Queensland, Brisbane, QLD, Australia
  2. Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom
  3. University of Auckland, Auckland, New Zealand
  4. AgResearch, Christchurch, New Zealand

ABC toxins are bacterially secreted pore-forming toxins of ~2 MDa in size, and are strongly implicated in insect and human pathogenicity. These multi-chain protein complexes have (at minimum) a tripartite architecture in which the A subunit specifically recognises the host cell membrane in the pre-pore conformation, and subsequently perforates the membrane to enable translocation of a cytotoxin (subunit C) through a pore domain. This mechanism of pore formation and toxin translocation is accompanied by substantial structural domain rearrangements and prior to now, it has remained unclear whether these mechanisms are shared across the ABC toxin family or with any other pore-forming toxins. Here we present cryo-EM structures of the Yersinia entomophaga toxin complex, YenTc, which is representative of a novel ABC toxin subtype with chitinase activity. Using a Tecnai F30 with K2 direct electron detector, we obtained cryo-EM maps of YenTc in different conformations using various solubilisation methods. We performed 3D refinements of the YenTc holotoxin (including subunits A, B and C and Chitinase 1 and 2) as well as focused refinements of dynamic and heterogeneous subunits. This resulted in 3D maps with resolutions varying (on average) from 4.2 Å to ~8 Å, which was sufficient for de novo model building and/or pseudo-atomic model fitting. Our models provide a comprehensive, 4D picture of YenTc and molecular drivers of its conformational changes: most notably the protrusion of a central α-helical translocation pore from the complex and the rearrangement of domains involved in receptor recognition, particularly the two exposed chitinases. Overall, our results suggest that ABC toxins combine a conserved pore-forming architecture with diverse motifs that impart specificity during cell recognition. This forms the basis for future applications of YenTc as a target-specific drug delivery machine.