Hexameric Modules Orchestrate the Hierarchical Pore Assembly of Cytolysin Resolved by Multistack Gel Electrophoresis and Structural Imaging

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins that assemble on target membranes through coordinated subunit interactions. Understanding the precise structure and assembly pathway of intermediate oligomeric complexes is crucial for controlling their function at biomolecular interfaces and for nanoscale membrane engineering. Using perfringolysin O (PFO) as a model system for protein assembly, we combine high-resolution atomic force microscopy (AFM) with multistack gel electrophoresis to resolve discrete hexameric modules and higher-order assemblies that are difficult to capture by conventional cryo-EM because of their transient flexibility. We show that the W165T mutant stabilizes a kinetically trapped hexamer, whereas wild-type PFO forms cooperative higher-order complexes through hierarchical hexamer-hexamer association. A kinetic model that combines both seeded monomer addition and interhexamer coupling recapitulates the observed population distributions and predicts how environmental factors, such as temperature and protein incorporation, modulate assembly efficiency by physicochemical parameters. Together, these findings identify modular hexamers as the building block, reveal a dominant, hierarchical pathway for PFO pore formation, and provide physical insight into protein assembly at interfaces, with applications for designing controlled nanoscale systems.