Perfringolysin O (PFO) belongs to the family of cholesterol-dependent cytolysins. PFO binding to cholesterol-containing membranes initiates series of structural changes in the protein which result in the formation of β-barrel pores, leading to cell lysis1 .
D4 domain is a C-terminal part of PFO responsible for recognition and binding to cholesterol. The crystallographic studies showed that D4 consists of two four-stranded β-sheets which create three short loops and one longer, conserved tryptophan rich loop (undecapeptide) composed of 11 amino acid residues (ECTGLAWEWWR)2 . At the beginning undecapeptide was regarded to be responsible for cholesterol recognition and binding. However, recent studies indicates that the tryptophan rich loop is also crucial for the pre-pore assembly and pre-pore to pore transition.3 4
Additionally, it was showed that mutation R468A decreased the ability of PFO binding to cholesterol and abolished the oligomer formation as a result of disruption of allosteric coupling between D4 and other domains4 .
In our studies we used hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) method in order to analyze the structural consequences of R468A mutation. This approach allows to monitor the dynamics and structural changes of protein in solution as well as incorporated into the membrane5 . The measurements of deuterium exchange rates in backbone hydrogens indicated that the PFOR468A mutant is highly dynamic - only several peptides have deuterium uptake lower than 50 %. Observed destabilization in all domains shows how a single change in amino acid chain localized in the undecapeptide can lead to a global effect in protein structure and dynamics.
This analysis explains disorder in the properties of PFOR468A - the deformed protein structure cannot result in correct oligomerization and proper pore formation. These results strongly suggest that C- terminal end plays a crucial role in stabilization of appropriate toxin structure.