Bell C E, Poon P H, Schumaker V N, Eisenberg D
UCLA-DOE Lab of Structural Biology and Molecular Medicine, Molecular Biology Institute, and Department of Chemistry and Biochemistry, Box 951569, University of California at Los Angeles, Los Angeles, California 90095-1569, USA.
Biochemistry. 1997 Dec 9;36(49):15201-7. doi: 10.1021/bi971301x.
Diphtheria toxin (DT) is a 58 kDa protein, secreted by lysogenic strains of Corynebacterium diphtheriae, that causes the disease diphtheria in humans. The catalytic (C) domain of DT kills host cells by gaining entry into the cytoplasm and inhibiting protein synthesis. The translocation of the C domain across the endosomal membrane and into the cytoplasm of a host cell is mediated by the translocation (T) domain of DT. This process is triggered by acidification from pH approximately 7 to pH approximately 5 within the endosome. Here we show that crm45 (cross-reacting material of 45 kDa), a 45 kDa deletion mutant of DT which contains the C and T domains but lacks the C-terminal receptor-binding (R) domain, undergoes a transition from a monomer to a large oligomer upon acidification from pH 7.0 to pH 5.0. Dynamic light scattering analysis of crm45 at pH 5.0 results in a polydispersity value of only 8-17%, suggesting that the oligomer is uniformly sized. Using analytical ultracentrifugation, measurements of the sedimentation rate and diffusion coefficient of crm45 at pH 5.0 result in a molecular mass determination of 890 +/- 40 kDa (20 +/- 1 subunits) for the oligomer. Equilibrium sedimentation data on crm45 at pH 5.0 are best fit by a single species with a mass of 1000 +/- 50 kDa (24 +/- 1 subunits). These results reveal the pH-dependent formation of a uniformly sized, 20-24 subunit oligomer of the C and T domains of DT, in solution. Because the oligomer of crm45 forms at the pH of the acidified endosome, it could be relevant to the translocation of the C domain of DT across the endosomal membrane and into the cytoplasm of host cells. The possible relevance of this oligomer of crm45 to the membrane translocation of the C domain of DT correlates with earlier kinetic studies of DT intoxication of Vero cells, which inferred the transfer of approximately 20 C domains of DT to the cytoplasm of host cells, in a single event.
白喉毒素（DT）是一种58 kDa的蛋白质，由产毒株白喉棒状杆菌分泌，可导致人类患白喉病。DT的催化（C）结构域通过进入细胞质并抑制蛋白质合成来杀死宿主细胞。C结构域穿过内体膜并进入宿主细胞细胞质的转运过程由DT的转运（T）结构域介导。这个过程是由内体中pH值从约7酸化到约5触发的。在这里，我们展示了crm45（45 kDa交叉反应物质），一种DT的45 kDa缺失突变体，它包含C和T结构域但缺少C末端受体结合（R）结构域，在pH值从7.0酸化到5.0时会从单体转变为大的寡聚体。对pH 5.0的crm45进行动态光散射分析，得到的多分散值仅为8 - 17%，这表明该寡聚体大小均匀。使用分析超速离心法，测量pH 5.0时crm45的沉降速率和扩散系数，得出该寡聚体的分子量为890±40 kDa（20±1个亚基）。pH 5.0时crm45的平衡沉降数据最适合由质量为1000±50 kDa（24±1个亚基）的单一物种来拟合。这些结果揭示了在溶液中DT的C和T结构域形成了大小均匀的、由20 - 24个亚基组成的寡聚体，且这种形成依赖于pH值。由于crm45的寡聚体在酸化内体的pH值下形成，它可能与DT的C结构域穿过内体膜并进入宿主细胞细胞质的转运过程有关。crm45的这种寡聚体与DT的C结构域的膜转运的可能相关性与早期对Vero细胞DT中毒的动力学研究相关，该研究推断在单个事件中约20个DT的C结构域转移到了宿主细胞的细胞质中。
