The mechanism by which the cholesterol-dependent cytolysins (CDCs) assemble their giant β-barrel pore in cholesterol-rich membranes has been the subject of intense study in the past two decades. A combination of structural, biophysical, and biochemical analyses has revealed deep insights into the series of complex and highly choreographed secondary and tertiary structural transitions that the CDCs undergo to assemble their β-barrel pore in eukaryotic membranes. Our knowledge of the molecular details of these dramatic structural changes in CDCs has transformed our understanding of how giant pore complexes are assembled and has been critical to our understanding of the mechanisms of other important classes of pore-forming toxins and proteins across the kingdoms of life. Finally, there are tantalizing hints that the CDC pore-forming mechanism is more sophisticated than previously imagined and that some CDCs are employed in pore-independent processes.

β-Barrel pore-forming toxins (βPFTs) form an obligatory oligomeric prepore intermediate before the formation of the β-barrel pore. The molecular components that control the critical prepore-to-pore transition remain unknown for βPFTs. Using the archetype βPFT perfringolysin O, we show that E183 of each monomer within the prepore complex forms an intermolecular electrostatic interaction with K336 of the adjacent monomer on completion of the prepore complex. The signal generated throughout the prepore complex by this interaction irrevocably commits it to the formation of the membrane-inserted giant β-barrel pore. This interaction supplies the free energy to overcome the energy barrier (determined here to be ∼19 kcal/mol) to the prepore-to-pore transition by the coordinated disruption of a critical interface within each monomer. These studies provide the first insight to our knowledge into the molecular mechanism that controls the prepore-to-pore transition for a βPFT.hemolysin | streptolysin | sterol | toxin | alpha-hemolysin T he cholesterol-dependent cytolysins (CDCs) make up the largest class of bacterial β-barrel pore-forming toxins (βPFTs) and are present in nearly 50 Gram-positive opportunistic pathogens. A central paradigm of βPFTs is the formation of an obligatory intermediate termed the prepore (1-6). The prepore is a membrane-bound oligomerized ring-shaped complex in which the membrane-spanning β-barrel pore has not formed. Pore conversion, characterized by β-barrel insertion, only occurs after prepore completion. For all βPFTs, the molecular mechanism or mechanisms that control the transition from the prepore to the pore remains unknown.The archetype CDC, perfringolysin O (PFO), coordinates the assembly of about 37 monomers into a prepore and then assembles and inserts into the membrane a β-barrel pore composed of 74 amphipathic hairpins (7-12). On prepore completion, a signal is generated within the oligomeric complex that triggers this transition. The nexus of the prepore-to-pore transition is the disruption of the interface that domain 3 (D3) forms with domains 1 and 2 (D1,2) (10) (Fig. 1). This interface is formed between D1,2 and one of the two D3 α-helical bundles. The D3 α-helical structures ultimately unfurl and refold into the two transmembrane β-hairpins (TMH1 and TMH2), which contribute to the formation of the giant β-barrel pore ( Fig. 1) (11)(12)(13). Pore formation also requires that the prepore undergo a 40-Å vertical collapse that allows the TMHs to span the bilayer (7,8,14).Here we show that the prepore-to-pore transition is controlled by an intermolecular electrostatic interaction between E183 and K336 in PFO. Loss of either charged residue traps PFO in a stable prepore state, whereas pore formation can be restored by weakening the D3-D1,2 interface by a single mutation or by increasing the temperature. Molecular measurements with cross-linkers and molecular dynamics simulations suggest these two residues are positioned to form a strong electrostatic interaction or salt bridge. Our stu...

Background:The cholesterol-dependent cytolysins (CDCs) have an identical cholesterol-binding motif but exhibit different binding parameters. Results: Binding affinity is altered by the loop three (L3) structure, which impacts pore-forming efficiency. Conclusion: The L3 structure affects its equilibrium between stabilizing (inserted) and destabilizing (uninserted) membrane interactions. Significance: The L3 structure provides CDCs with cellular selectivity by discriminating lipid environments surrounding membrane cholesterol.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.

The CbpA peptide-L460D pneumolysoid fusion protein was broadly protective against pneumococcal infection, with the potential for additional protection against other meningeal pathogens.