Perfringolysin O (PFO) is a toxic protein that forms β-barrel transmembrane pores upon binding to cholesterol-containing membranes. The formation of lytic pores requires conformational changes in PFO that lead to the conversion of water-soluble monomers into membrane-bound oligomers. Although the general outline of stepwise pore formation has been established, the underlying mechanistic details await clarification. To extend our understanding of the molecular mechanisms that control the pore formation, we compared the hydrogen-deuterium exchange patterns of PFO with its derivatives bearing mutations in the D3 domain. In the case of two of these mutations F318A, Y181A, known from previous work to lead to a decreased lytic activity, global destabilization of all protein domains was observed in their water-soluble forms. This was accompanied by local changes in D3 β-sheet, including unexpected stabilization of functionally important β1 strand in Y181A. In case of the double mutation (F318A/Y181A) that completely abolished the lytic activity, several local changes were retained, but the global destabilization effects of single mutations were reverted and hydrogen-deuterium exchange (HDX) pattern returned to PFO level. Strong structural perturbations were not observed in case of remaining variants in which other residues of the hydrophobic core of D3 domain were substituted by alanine. Our results indicate the existence in PFO of a well-tuned H-bonding network that maintains the stability of the D3 β-strands at appropriate level at each transformation step. F318 and Y181 moieties participate in this network and their role extends beyond their direct intermolecular interaction during oligomerization that was identified previously.
Keywords: Cholesterol-dependent cytolysin; Hydrogen-deuterium exchange; Oligomerization; Pore formation; Structure.
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