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Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit β-strand alignment

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Abstract

Cholesterol-dependent cytolysins are bacterial protein toxins that bind to cholesterol-containing membranes, form oligomeric complexes and insert into the bilayer to create large aqueous pores. Membrane-dependent structural rearrangements required to initiate the oligomerization of perfringolysin O monomers have been identified, as have the monomer-monomer interaction surfaces, using site-specific mutagenesis, disulfide trapping and multiple fluorescence techniques. Upon binding to the membrane, a structural element in perfringolysin O moves to expose the edge of a previously hidden β-strand that forms the monomer-monomer interface and is required for oligomer assembly. The β-strands that form the interface each contain a single aromatic residue, and these aromatics appear to stack, thereby aligning the transmembrane β-hairpins of adjacent monomers in the proper register for insertion. Collectively, these data reveal a novel membrane binding–dependent mechanism for regulating cytolysin monomer-monomer association and pore formation.

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Figure 1: PFO structure, structural elements and mutations.
Figure 2: Effect of β4-β5 disulfide-locking on pore formation.
Figure 3: D4-membrane interaction triggers a conformational rearrangement around β4 in D3.
Figure 4: Mutation of glycines at the end of β4 blocks oligomerization.
Figure 5: Excimer formation detected by pyrenes attached to β1 and β4 of neighboring monomers.
Figure 7: Proper relative positioning of aromatic residues in β1 and β4 is critical for pore formation.
Figure 6: Aromatic residue involvement in PFO function.
Figure 8: Mechanism of oligomerization and intermolecular β-sheet formation in PFO.

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Acknowledgements

This work was supported by US National Institutes of Health grant AI 37657 and the Robert A. Welch Foundation.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Texas A&M University, College Station, 77843, Texas, USA

    Rajesh Ramachandran & Arthur E Johnson

  2. Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, 73104, Oklahoma, USA

    Rodney K Tweten

  3. Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, 77843-1114, Texas, USA

    Arthur E Johnson

  4. Department of Chemistry, Texas A&M University, College Station, 77843, Texas, USA

    Arthur E Johnson

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  1. Rajesh Ramachandran
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  2. Rodney K Tweten
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Correspondence to Arthur E Johnson.

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Ramachandran, R., Tweten, R. & Johnson, A. Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit β-strand alignment. Nat Struct Mol Biol 11, 697–705 (2004). https://doi.org/10.1038/nsmb793

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