Poster Presentation The 46th Lorne Conference on Protein Structure and Function 2021

Understanding the formation of a novel two-component membrane pore by pore-forming toxins structurally related to cholesterol-dependent cytolysins (#219)

Bronte A. Johnstone 1 , Sara L. Lawrence 2 , Craig J. Morton 1 , Jordan C. Evans 3 , Michelle P. Christie 1 , Rodney K. Tweten 3 , Michael W. Parker 1 2
  1. Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Victoria, Australia
  2. ACRF Rational Drug Discovery Centre, St Vincent's Institute of Medical Research, Fitzroy, VIC, Australia
  3. Department of Microbiology and Immunology, University of Oklahoma, Oklahoma City, OK 73104, USA

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins that are secreted as soluble monomers and oligomerise into large circular pre-pores on the surface of cholesterol-rich membranes. Various structural changes and transitions results in insertion of β-hairpins into the lipid bilayer, forming a large β-barrel pore that results in cell lysis [1]. We have identified a highly conserved structural motif of CDCs that plays a critical role in the prepore-to-pore transition. Furthermore, this motif is also highly conserved in a large, diverse family of uncharacterised proteins from over 220 species, which we have designated the name “CDC-like” (CDCL) proteins [2]. Many of these CDCLs exist as homologous pairs. We have identified a novel CDCL pair, referred to as ALY long (ALYL) and ALY short (ALYS), that originates from the species Elizabethkingia anophelis, a commensal bacterium of the Anopheles mosquito. X-ray crystallography revealed the structure of monomeric ALYL consists of characteristic CDC domain 1 – 3 structure; however, domain 4 differs from that of CDCs significantly. In the presence of lipids, ALYL and ALYS show weak pore-forming activity and analysis by negative-staining electron microscopy reveals a large circular oligomeric complex reminiscent of CDC pore complexes. ALYalso forms a non-lytic circular oligomer in the absence of ALYL. XL-MS data reveals an initial insight into the structural changes between the monomeric and protomeric states, which will be further supported by the high-resolution cryo-EM structure of the complex. In summary, we have shown that the ALY toxins share some structural resemblance to CDCs, but form a two-component pore complex that is unique to the CDC family. This study establishes the beginning of an investigation into the large family of novel CDC-like proteins present in a wide range of bacterial species and are suspected to play key roles in microbial survival and human disease.

  1. [1] Christie, M.P., Johnstone, B.A., Tweten, R.K. et al. Cholesterol-dependent cytolysins: from water-soluble state to membrane pore. Biophys Rev 10, 1337–1348 (2018). https://doi.org/10.1007/s12551-018-0448-x
  2. [2] Evans, J.C., Johnstone, B.A., Lawrence, S.L., Morton C.J., Christie, M.P., Parker, M.W., and Tweten, R.K. A Key Motif in the Cholesterol-Dependent Cytolysins Reveals a Large Family of Related Proteins. mBio (2020), 11 (5) e02351-20; DOI: 10.1128/mBio.02351-20