Perfringolysin O (PFO) belongs to the family of cholesterol-dependent cytolysins. Upon binding to a cholesterol-containing membrane, PFO undergoes a series of structural changes that result in the formation of a β-barrel pore and cell lysis. Recognition and binding to cholesterol are mediated by the D4 domain, one of four domains of PFO. The D4 domain contains a conserved tryptophan-rich loop named undecapeptide (ECTGLAWEWWR) in which arginine 468 is essential for retaining allosteric coupling between D4 and other domains during interaction of PFO with the membrane. In this report we studied the impact of R468A mutation on the whole protein structure using hydrogen-deuterium exchange coupled with mass spectrometry. We found that in aqueous solution, compared to wild type (PFO), PFO showed increased deuterium uptake due to exposure of internal toxin regions to the solvent. This change reflected an overall structural destabilization of PFO in solution. Conversely, upon binding to cholesterol-containing membranes, PFO revealed a profound decrease of hydrogen-deuterium exchange when compared to PFO. This block of deuterium uptake resulted from PFO-induced aggregation and fusion of liposomes, as found by dynamic light scattering, microscopic observations and FRET measurements. In the result of liposome aggregation and fusion, the entire PFO molecule became shielded from aqueous solution and thereby was protected against proteolytic digestion and deuteration. We have established that structural changes induced by the R468A mutation lead to exposure of an additional cholesterol-independent liposome-binding site in PFO that confers its fusogenic property, altering the mode of the toxin action.