Gazit E, Burshtein N, Ellar D J, Sawyer T, Shai Y
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel.
Biochemistry. 1997 Dec 9;36(49):15546-54. doi: 10.1021/bi9707584.
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.
CytA毒素通过在靶细胞膜内形成孔道来发挥其活性。然而，孔道形成的确切机制以及参与毒性活性的结构元件尚未确定。最近，高度相似的CytB毒素的结构得到了解析（Li等人，1996年），有人提出β桶可能是孔道的结构。由于毒素之间的相似性，通过序列比对预测了CytA中α螺旋和β折叠的存在和位置。在此，合成了与β5、β6和β7链、CytA毒素的保守非螺旋区域（P149 - 170）、螺旋B和D以及螺旋A的类似物相对应的肽段，进行荧光标记并进行表征。我们发现，与螺旋A和C不同（Gazit和Shai，1993年），β链肽段和螺旋B都不能与脂质膜相互作用，而P149 - 170和螺旋D与膜的结合较弱。膜渗透实验表明，CytA毒素通过几个单体的聚集来发挥其活性。为了了解可能介导CytA寡聚化的结构元件，研究了合成肽与膜结合的CytA相互作用的能力。螺旋A和C，但不是β链、螺旋D或对照肽，导致膜结合的荧光素标记的CytA的荧光大幅增加，而螺旋B只有轻微影响。此外，添加重排的螺旋A，一种与螺旋A组成相同但只有两对氨基酸重排的肽段，并不影响荧光。添加未标记的CytA也导致荧光强度增加，进一步证明了膜内CytA单体之间的相互作用。综上所述，我们的结果为CytA毒素在膜内自组装以及螺旋A和C是参与毒素膜相互作用和分子间组装的主要结构元件这一观点提供了进一步支持。
