Silverman J A, Mindell J A, Finkelstein A, Shen W H, Collier R J
Department of Microbiology and Molecular Genetics, Shipley Institute of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1994 Sep 9;269(36):22524-32.
Entry of the catalytic domain of diphtheria toxin into the cytoplasma of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH. To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain. Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K. Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin. Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells > 100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction. All three mutations also altered the pH-dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane. E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5. Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain. A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action. The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model. The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain.
白喉毒素催化结构域进入真核细胞的细胞质取决于T（跨膜）结构域插入内体膜，这一过程由低pH触发。为了探究插入机制，我们对内毒素T结构域螺旋发夹区域内的可电离残基进行了突变。只有三个突变对细胞毒性产生了显著影响，即D295K、E349K和D352K。这些突变均代表螺旋发夹顶端酸性残基被碱性残基取代。在TH8/9发夹中，用赖氨酸取代谷氨酸349或天冬氨酸352，对Vero细胞的毒性降低了100倍以上，而在TH5/6/7发夹中的三个酸性残基之一用赖氨酸取代天冬氨酸295，其毒性降低幅度较小。这三个突变还改变了毒素在人工双层膜或质膜中形成的通道的pH依赖性形成和/或离子电导。E349K或D352K并未改变毒素在pH 5附近发生的构象变化的pH依赖性。我们的研究结果支持这样的假设，即TH8/9发夹在低pH介导的T结构域部分展开后插入内体膜。该发夹顶端的一个正性残基显然会抑制插入并阻断毒素作用。其他地方描述的选定突变体形成的通道的离子传导特性与该模型一致。T结构域整合膜形式中TH5/6/7发夹的状态仍不确定。
