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Bacillus thuringiensis Cytolytic Toxin Associates Specifically with Its Synthetic Helices A and C in the Membrane Bound State. Implications for the Assembly of Oligomeric Transmembrane Pores

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    Bacillus thuringiensis Cytolytic Toxin Associates Specifically with Its Synthetic Helices A and C in the Membrane Bound State. Implications for the Assembly of Oligomeric Transmembrane Pores†
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    Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel, and Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, U.K.
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    Biochemistry

    Cite this: Biochemistry 1997, 36, 49, 15546–15554
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    https://doi.org/10.1021/bi9707584
    Published December 9, 1997
    Copyright © 1997 American Chemical Society
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    Abstract

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    The CytA toxin exerts its activity by the formation of pores within target cell membranes. However, the exact mechanism of pore formation and the structural elements that are involved in the toxic activity are yet to be determined. Recently, the structure of the highly similar CytB toxin was solved (Li et al., 1996), and a β-barrel was suggested as a possible structure of the pores. Due to the similarity between the toxins, the existence and positioning of α-helices and β-sheets in CytA were predicted from the alignment of the sequences. Here peptides corresponding to β5, β6, and β7 strands, to a conserved nonhelical region of the CytA toxin (P149-170), to helices B and D, and to an analogue of helix A were synthesized, fluorescently labeled, and characterized. We found that, unlike helices A and C (Gazit and Shai, 1993), neither the β-strand peptides nor helix B could interact with lipid membranes, whereas P149-170 and helix D bind the membrane weakly. Membrane permeation experiments suggested that CytA toxin exerts its activity by aggregation of several monomers. To learn about the structural elements that may mediate CytA oligomerization, the ability of the synthetic peptides to interact with membrane-bound CytA was studied. Helices A and C, but not the β-strands, helix D, or a control peptide, caused a large increase in the fluorescence of membrane-bound fluorescein-labeled CytA, whereas helix B had only a slight effect. Moreover, the addition of rearranged helix A, a peptide with the same composition as helix A, but with only two pairs of amino acids rearranged, did not affect the fluorescence. The addition of unlabeled CytA also caused an increase in the fluorescence intensity, further demonstrating the interaction between CytA monomers within the membrane. Taken together, our results provide further support for the suggestion that the CytA toxin self-assembles within membrane and that helices A and C are major structural elements involved in the membrane interaction and intermolecular assembly of the toxin.

    Copyright © 1997 American Chemical Society

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    †

     This research was supported in part by the Basic Research Foundation administered by the Israel Academy of Sciences and Humanities. E.G. is a recipient of a doctoral fellowship from the Clore Foundation Scholar Program.

    ‡

     Weizmann Institute of Science.

    §

     University of Cambridge.

    *

     To whom correspondence should be addressed. Tel:  +972-8-9342711. Fax:  +972-8-9344112. E-mail:  [email protected]. ac.il.

    ✗

     Abstract published in Advance ACS Abstracts, November 15, 1997.

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    Biochemistry

    Cite this: Biochemistry 1997, 36, 49, 15546–15554
    Click to copy citationCitation copied!
    https://doi.org/10.1021/bi9707584
    Published December 9, 1997
    Copyright © 1997 American Chemical Society
    Request reuse permissions

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