Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Structural studies of recently identified Bacteroides fragilis Cholesterol-dependent Cytolysin Like (CDCL) proteins (#102)

Riya Joseph 1 2 , Bronte A Johnstone 1 2 , Michelle P Christie 1 2 , Craig J Morton 1 3 , Rodney K Tweten 4 , Michael W Parker 1 2 5
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria,, Australia
  2. ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, Victoria, Australia
  3. CSIRO Biomedical Manufacturing Program, Clayton, Victoria, Australia
  4. Department of Microbiology and Immunology, University of Oklahoma, Health Sciences Center, Oklahoma City, Oklahoma , USA
  5. Australian Cancer Research Foundation Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy , Victoria, Australia

Pore-forming proteins (PFPs) are a large family of proteins capable of punching holes in their target cell membranes. Cholesterol-dependent cytolysins (CDCs) are a prominent class of bacterial PFPs. Although these proteins have been studied for the past four decades, we still don’t clearly understand the pore formation mechanism of these proteins, especially how these proteins are inserted into their target membranes (1).

The recent identification of cholesterol-dependent cytolysin-like proteins (CDCLs), a novel, diverse family of over 300 uncharacterised proteins, led to a further understanding of the pore-forming mechanism of CDCs. CDCLs and CDCs share a common motif (F/Y-F/Y-Xn-YGR) and show high structural similarity, particularly in the D1, D2 and D3 domains (2). Interestingly, the D4 domain of the CDCLs is very different from that of CDCs, and in some CDCLs, the entire C-terminal D4 domain is absent. Those containing domains 1-4 are referred to as long CDCLs, while those that don’t have a D4 domain are referred to as short CDCLs.

Bacteroides fragilis are gram-negative anaerobic bacteria found in the human colon (3). We are currently investigating the structure and pore-forming activity of a pair of long and short CDCLs from B. fragilis. Previous studies of Elizabethkingia anopheles CDCLs showed that both short and long forms are required for active pore formation, making it a two-component pore system (2).

We aim to study the stoichiometry of B. fragilis CDCLs in their pore state and determine the distinct steps involved in pore formation using X-ray crystallography for individual monomeric states, cryo-electron microscopy for the pore complex and small-angle X-ray scattering for solution structures. Progress towards these aims will be presented. This study will help discover the similarities and differences in how different CDCLs punch holes in their target cell membranes and reveal how different D4 domains influence pore formation.

  1. Johnstone BA, Joseph R, Christie MP, Morton CJ, McGuiness C, Walsh JC, et al. Cholesterol-dependent cytolysins: The outstanding questions. IUBMB Life. 2022. 2. Evans JC, Johnstone BA, Lawrence SL, Morton CJ, Christie MP, Parker MW, et al. A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins. mBio. 2020;11(5). 3. Wexler HM. Bacteroides: the good, the bad, and the nitty-gritty. Clin Microbiol Rev. 2007;20(4):593-621.