Pathogenic bacteria secrete pore-forming toxins (PFTs) to attack target cells. PFTs are expressed as water-soluble monomeric proteins, which oligomerize into nonlytic prepore intermediates on the target cell membrane before forming membrane-spanning pores. Despite a wealth of biochemical data, the lack of high-resolution prepore structural information has hampered understanding of the b-barrel formation process. Here, we report crystal structures of staphylococcal g-haemolysin and leucocidin prepores. The structures reveal a disordered bottom half of the b-barrel corresponding to the transmembrane region, and a rigid upper extramembrane half. Spectroscopic analysis of fluorescently labelled mutants confirmed that the prepore is distinct from the pore within the transmembrane region. Mutational analysis also indicates a pivotal role for the glycine residue located at the lipid-solvent interface as a 'joint' between the two halves of the b-barrel. These observations suggest a two-step transmembrane b-barrel pore formation mechanism in which the upper extramembrane and bottom transmembrane regions are formed independently.

16Staphylococcal α-hemolysin (α-HL) is a β-barrel pore-forming toxin (PFT) expressed by 17 Staphylococcus aureus. α-HL is secreted as a water-soluble monomeric protein, which binds to target 18 membranes and forms membrane-inserted heptameric pores. To explore the pore-forming 19 mechanism of α-HL in detail, we determined the crystal structure of the α-HL monomer and prepore 20 using H35A mutant and W179A/R200A mutant, respectively. Although the overall structure of the 21 monomer was similar to that of other staphylococcal PFTs, a marked difference was observed in the Based on these observations, we proposed a dynamic molecular mechanism of pore formation for 41l The prestem is fastened by a key hydrogen bond between Asp45 and Tyr118 in monomer. 42l In prepore, the transmembrane region is roughly formed with flexibility. 43l Upon oligomerization, the released amino latch destroys the key interaction to release prestem. 44l A dynamic pore-forming mechanism, where the amino latch plays a key role, is proposed.

Staphylococcal -haemolysin is a -barrel pore-forming toxin expressed by Staphylococcus aureus. -Haemolysin is secreted as a water-soluble monomeric protein which binds to target membranes and forms membrane-inserted heptameric pores. Although the crystal structures of the heptameric pore and monomer bound to an antibody have been determined, that of monomeric -haemolysin without binder has yet to be elucidated. Previous mutation studies showed that mutants of His35 retain the monomeric structure but are unable to assemble into heptamers. Here, -haemolysin H35A mutants were expressed, purified and crystallized. Diffraction data were collected to 2.90 Å resolution. The crystals belonged to space group P6 1 , with unit-cell parameters a = b = 151.3, c = 145.0 Å . Molecular replacement found four molecules in an asymmetric unit. The relative orientation among molecules was distinct from that of the pore, indicating that the crystal contained monomeric -haemolysin.

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