Oral Presentation (15 min) The 48th Lorne Conference on Protein Structure and Function 2023

Using serial femtosecond crystallography to solve the structures of pesticidal proteins from natural crystals (#49)

Lainey J Williamson 1 , Marina Galchenkova 2 , Hannah L Best 1 , Richard J Bean 3 , Anna Munke 2 , Salah Awel 2 , Gisel Pena 2 , Juraj Knoska 2 , Robin Schubert 3 , Katerina Doerner 3 , Joana Valerio 3 , Emyr Lloyd-Evans 1 , Marco Kloos 3 , Marcin Sikorski 3 , Grant Mills 3 , Johan Bieleki 3 , Henry Kirkwood 3 , Chan Kim 3 , Raphael de Wijn 3 , Kristina Lorenzen 3 , P. Lourdu Xavier 2 4 , Aida Rahmani 2 , Luca Gelisio 2 , Oleksandr Yefanov 2 , Adrian P Mancuso 3 5 , Henry N Chapman 2 6 7 , Pierre J Rizkallah 8 , Dominik Oberthür 2 , Neil Crickmore 9 , Colin Berry 1 , Hyun-Woo Park 10 , Dennis K Bideshi 10 , Alesandra Henkel 2 , Viviane Kremling 2 , Bjarne Klopprogge 2 , Mark Young 1 , Brian Federici 11
  1. School of Biosciences, Cardiff University, Cardiff, United Kingdom
  2. Center for Free Electron Laser Science CFEL, Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany
  3. European XFEL GmbH, Hamburg, Germany
  4. Max-Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany
  5. Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia
  6. Centre for Ultrafast Imaging, Universität Hamburg, Hamburg, Germany
  7. Department of Physics, Universität Hamburg, Hamburg, Germany
  8. School of Medicine, Cardiff University, Cardiff, United Kingdom
  9. School of Life Sciences, University of Sussex, Falmer, UK
  10. Department of Biological Sciences, California Baptist University, California, USA
  11. Department of Entomology and Institute for Integrative Genome Biology, , University of California, California, USA

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are gram-positive, spore-forming bacteria that produce several classes of structurally diverse pesticidal proteins, many of which are produced naturally as crystalline inclusions.  These proteins constitute the major factors in bioinsecticides and transgenic Bt crops.  The general mode of action begins with dissolution of the pesticidal crystal proteins in the acidic or alkaline environment of the target insect larvae gut, followed by proteolytic processing to the active form.  The activated protein oligomerizes and binds specifically to receptors present on the midgut epithelial cells, leading to pore-formation, cell lysis, and ultimately, insect death.  While some protein structures are available, many are uncharacterised, particularly in the protoxin forms.  In addition, there is currently a limited understanding surrounding the mechanism of natural crystallisation and the processes contributing to subsequent dissolution.  Here, we utilised serial femtosecond crystallography (SFX) at the European X-ray free electron laser (XFEL) facility to determine the structures of pesticidal proteins from natural crystals isolated from spores.  

SFX was employed to solve structures including Tpp49Aa1 and two forms of Cry8Ba2 produced naturally in the same preparation.  The Cry8Ba2 structure represents the first structure of a non-mutated Cry toxin in the long protoxin form and sheds light on the arrangement of Cry toxins into natural crystals.  In addition, mixing experiments conducted at varied pH enabled investigations of the early events leading up to the dissolution of natural Tpp49Aa1 crystals.  This work will pave the way for further investigations on the structure and dynamics of bacterial insecticides, with the goal of developing better and greener insecticides for agricultural pest-control.