Poster Presentation The 3rd Prato Conference on Pore Forming Proteins 2015

Examination of structural dynamics of perfringolysin O at pre-pore state using hydrogen-deuterium exchange coupled with mass spectrometry (#44)

Aleksandra Kacprzyk-Stokowiec 1 , Magdalena Kulma 1 , Gabriela Traczyk 2 , Katarzyna Kwiatkowska 2 , Michal Dadlez 1
  1. Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland
  2. Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

Perfringolysin O (PFO) is a toxic protein produced and secreted by Clostridium perfringens. It is a member of a large group of cholesterol-dependent cytolysin (CDC), that interfere with biological membranes with high cholesterol content. The interaction of PFO with lipid bilayer starts when the protein recognizes cholesterol and binds to the surface of the membrane. Next PFO forms pre-oligomers, that transform into oligomers which eventually cause creation of transmembrane pore. Although the general series of steps of PFO action is known, a detailed data about toxin structure at particular stage requires investigation. Previously, using a hydrogen-deuterium exchange method coupled with mass spectrometry (HDX-MS), we showed that the proper pore formation (last stage) depends on stabilization of D1 domain of perfringolysin O 1. In our current experiments we examined PFO derivatives carrying the mutations in β1 (Y181A) and β4 (F318A) strands which are responsible for monomers interaction. This proteins were expected to form only oligomeric pre-pores on the surface of the lipid bilayers and not to create mature pores 2 3. First, the lytic activity and cholesterol binding specificity of mutated proteins were analyzed. Than we applied the HDX-MS technique to uncover the regions of PFO which undergo structural changes in pre-pore stage. Those results together with our previous finding bring us even closer than before to understanding of mechanism of perfringolysin O toxic pore formation. 

  1. Kacprzyk-Stokowiec A, Kulma M, Traczyk G, Kwiatkowska K, Sobota A, Dadlez M. (2014) Crucial role of perfringolysin O D1 domain in orchestrating structural transitions leading to membrane-perforating pores: a hydrogen-deuterium exchange study. J Biol Chem. 289(41):28738-52.
  2. Ramachandran R, Tweten RK, Johnson AE. (2005) The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation. PNAS; 102(20):7139-7144.
  3. Hotze EM, Heuck AP, Czajkowsky DM, Shao Z, Johnson AE, Tweten RK. (2002) Monomer-monomer interactions drive the prepore to pore conversion of a beta-barrel-forming cholesterol-dependent cytolysin. J Biol Chem.; 277(13):11597-605.