Next, a double mutant of PLY (PLY-DM) was produced with mutations of two critical amino acids (T459 and L460) within the cholesterol binding domain. When tested on sheep erythrocytes, PLY-DM was inactive, as expected for deleted cholesterol binding. In the BLI binding assay, however, PB-2 was still able to bind the PLY-DM mutant (Supplementary Figure ). Considering the necessity of the olefinic double bond for the inhibitory activity of PB compounds and the potential reactivity of this as a Michael acceptor, an alanine-mutant of the single cysteine residue, PLY-C428A, was generated and expressed. PLY-C428A was active both in the hemolysis and in the LDH assays and displayed similar cellular toxicity as the wildtype PLY. Removal of the cysteine residue in PLY-C428A reduced the potency of PB-3 resulting in an elevated IC50 of 11 µM in the hemolysis assay. Accordingly, higher concentrations of PB-3 were required for the protection of lung cells against this mutant compared to wildtype PLY (Supplementary Figure , Figure ). Formation and stability of the protein-ligand complex was investigated by protein mass spectrometry. Samples of wildtype PLY and PLY-C428A with and without PB-3 were analyzed by using native and denaturing protein MS conditions. Following exhaustive dialysis into ammonium acetate buffer, PLY showed native MS spectra, however, with numerous additional masses suggesting that MOPS buffer salts and glycerol still were attached to the protein (Supplementary Figure ). Moreover, dialyzed samples of both proteins in ammonium acetate buffer were instable and underwent rapid precipitation. In contrast, mass spectra of both proteins under denaturing conditions in acetonitrile-water could be recorded and deconvoluted reliably. A 1:1 mixture of PB-3 with wildtype PLY (6 µM each) delivered a broad protein mass peak reaching from the protein mass to the protein mass plus inhibitor PB-3 (Supplementary Figure ). This broad peak indicates that the protein-ligand complex was formed and was mostly stable in acetonitrile-water solution and decomposed partially during acceleration of molecular ions in the mass spectrometer resulting mass ions observed between the protein mass and the mass of the protein-ligand complex.