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Disulfide-bond scanning reveals assembly state and β-strand tilt angle of the PFO β-barrel

Nature Chemical Biology volume 9, pages 383–389 (2013)Cite this article

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Abstract

Perfringolysin O (PFO), a bacterial cholesterol-dependent cytolysin, binds a mammalian cell membrane, oligomerizes into a circular prepore complex (PPC) and forms a 250-Å transmembrane β-barrel pore in the cell membrane. Each PFO monomer has two sets of three short α-helices that unfold and ultimately refold into two transmembrane β-hairpin (TMH) components of the membrane-embedded β-barrel. Interstrand disulfide-bond scanning revealed that β-strands in a fully assembled PFO β-barrel were strictly aligned and tilted at 20° to the membrane perpendicular. In contrast, in a low temperature–trapped PPC intermediate, the TMHs were unfolded and had sufficient freedom of motion to interact transiently with each other, yet the TMHs were not aligned or stably hydrogen bonded. The PFO PPC-to-pore transition therefore converts TMHs in a dynamic folding intermediate far above the membrane into TMHs that are hydrogen bonded to those of adjacent subunits in the bilayer-embedded β-barrel.

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Figure 1: PFO structure and structural alterations.
Figure 2: Detection of disulfide-bonded PFO dimers in PPC and pore complexes.
Figure 3: β4-β1 crosslinking and β-strand alignment in pore complex.
Figure 4: Dimer formation in PPCs.
Figure 5: Fluorescence-detected changes in TMH environment reveal stages in TMH unfolding and alignment during pore formation.

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Acknowledgements

Support was provided by US National Institutes of Health grant AI-37657 (R.K.T.) and Robert A. Welch Foundation Chair grant BE-0017 (A.E.J.).

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Authors and Affiliations

  1. Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA

    Takehiro K Sato & Arthur E Johnson

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

    Rodney K Tweten

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

    Arthur E Johnson

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

    Arthur E Johnson

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  1. Takehiro K Sato
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T.K.S. did experiments and contributed to experimental design and writing text, and A.E.J. and R.K.T. contributed to experimental design and writing text.

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Correspondence to Arthur E Johnson.

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Sato, T., Tweten, R. & Johnson, A. Disulfide-bond scanning reveals assembly state and β-strand tilt angle of the PFO β-barrel. Nat Chem Biol 9, 383–389 (2013). https://doi.org/10.1038/nchembio.1228

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