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.
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 beta-barrel was suggested as a possible structure of the pores. Due to the similarity between the toxins, the existence and positioning of alpha-helices and beta-sheets in CytA were predicted from the alignment of the sequences. Here peptides corresponding to beta5, beta6, and beta7 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 beta-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 beta-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.
Gazit
E
E
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel.
Burshtein
N
N
Ellar
D J
DJ
Sawyer
T
T
Shai
Y
Y
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D002942:Circular Dichroism; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D006460:Hemolysin Proteins; D006461:Hemolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis