Step-by-Step Maturation Mechanism of Binary Toxin Pore Revealed by Cryo-EM Analysis

Membrane pore-forming proteins (PFPs) form ring-shaped membrane-translocating oligomers on membranes, contributing to infection, immunity, and cell death functions. Binary toxins produced by some bacteria consist of an enzymatic component that acts as a toxin and a membrane-binding component that forms a pore that delivers the enzymatic component into target cells. Cryo-electron microscopy (cryo-EM) has advanced our understanding of these translocation mechanisms by revealing several binary toxin complexes structures. However, the mechanisms underlying the initial pore formation remain unclear. We determined the structures of several oligomeric forms of the membrane-binding component Ib of the Iota toxin from Clostridium perfringens at various stages of pore formation. Structural comparisons revealed how the symmetrically arranged soluble oligomer (prepore) asymmetrically mature into a transmembrane oligomer (pore). These findings enhance our understanding of mechanisms of PFP and provide a structural basis for developing nanodevices using membrane pores. Significance StatementAlthough the mode of action of pore-forming proteins (PFPs) has been increasingly elucidated, the structural mechanisms underlying their maturation into membrane-spanning pores remain elusive. One of the key challenges is the difficulty in capturing the intermediate states between the soluble prepore and the mature pore, and its structural analysis. In this study, we successfully prepared the super-complex formed by a bacterial toxin PFP. The super-complex inhibited the constituting oligomers to maturate into the pore, which enabled structural determination of multiple intermediate state. Comparative structural analysis revealed that the transition from prepore to pore occurs through stepwise, domino-like conformational changes. The analysis workflow and insights from this study are expected to advance the field of PFP research.