Abstract
Ostreolysin A6 (OlyA6) is an oyster mushroom-derived membrane-binding protein that, upon recruitment of its partner protein, pleurotolysin B, forms a cytolytic membrane pore complex. OlyA6 itself is not cytolytic but has been reported to exhibit pro-apoptotic activities in cell culture. Here we report the formation dynamics and the structure of OlyA6 assembly on a lipid membrane containing an OlyA6 high-affinity receptor, ceramide phosphoethanolamine, and cholesterol. High-speed atomic force microscopy revealed the reorganization of OlyA6 dimers from initial random surface coverage to 2D protein crystals composed of hexameric OlyA6 repeat units. Crystal growth took place predominantly in the longitudinal direction by the association of OlyA6 dimers, forming a hexameric unit cell. Molecular-level examination of the OlyA6 crystal elucidated the arrangement of dimers within the unit cell and the structure of the dimer that recruits pleurotolysin B for pore formation.