Characteristic interaction surfaces of biological molecules have evolved through modifications of genetic material to control physiological processes through biomolecular interactions. Biological membranes represent a large interaction platform for many proteins. Peripheral membrane proteins can specifically recognize membrane lipids and are often involved in attack and defense mechanisms. Toxins from the family of cholesterol-dependent cytolysins represent a large group of virulence factors of Gram-positive bacteria, the archetypal representative of which is perfringolizine O from the bacterium Clostridium perfringens. The initial contact of the water-soluble toxin monomers is enabled by amino acids in the loops of the binding domain, which act as determinants of cholesterol specificity, although the exact
mechanism of membrane cholesterol recognition remains to be elucidated. The biochemical diversity of the interaction sites has also not yet been described. Using the in vitro evolution approach with ribosome display and
lipid vesicles of different composition, we investigated the biochemical and biophysical diversity of the perfringolysin O protein interaction surface. Affinity selection for cholesterol-rich vesicles mainly enriched amino acids
identical or chemically similar to naturally conserved amino acids. We also identified rare variants representing an alternative evolutionary pathway. No unique amino acid enrichment was detected after affinity selection for
cholesterol-free vesicles, suggesting that the binding domain of the toxin is evolutionarily adapted to specifically recognize membrane cholesterol and that amino acid substitutions in the contact surface cannot alter the specificity of the protein.