Institut für Biologie, FG Mikrobiologie, Universität Kassel, Kassel, Germany.
Toxins (Basel). 2013 May 3;5(5):958-68. doi: 10.3390/toxins5050958.
Diphtheria toxin (DT) inhibits eukaryotic translation elongation factor 2 (eEF2) by ADP-ribosylation in a fashion that requires diphthamide, a modified histidine residue on eEF2. In budding yeast, diphthamide formation involves seven genes, DPH1-DPH7. In an effort to further study diphthamide synthesis and interrelation among the Dph proteins, we found, by expression in E. coli and co-immune precipitation in yeast, that Dph1 and Dph2 interact and that they form a complex with Dph3. Protein-protein interaction mapping shows that Dph1-Dph3 complex formation can be dissected by progressive DPH1 gene truncations. This identifies N- and C-terminal domains on Dph1 that are crucial for diphthamide synthesis, DT action and cytotoxicity of sordarin, another microbial eEF2 inhibitor. Intriguingly, dph1 truncation mutants are sensitive to overexpression of DPH5, the gene necessary to synthesize diphthine from the first diphthamide pathway intermediate produced by Dph1-Dph3. This is in stark contrast to dph6 mutants, which also lack the ability to form diphthamide but are resistant to growth inhibition by excess Dph5 levels. As judged from site-specific mutagenesis, the amidation reaction itself relies on a conserved ATP binding domain in Dph6 that, when altered, blocks diphthamide formation and confers resistance to eEF2 inhibition by sordarin.
白喉毒素 (DT) 通过 ADP-核糖基化抑制真核翻译延伸因子 2 (eEF2)，这种修饰需要依赖于 eEF2 上的一种修饰组氨酸残基二氢喋呤。在芽殖酵母中，二氢喋呤的形成涉及七个基因，DPH1-DPH7。为了进一步研究二氢喋呤的合成以及 Dph 蛋白之间的相互关系，我们通过在大肠杆菌中的表达和在酵母中的共免疫沉淀发现，Dph1 和 Dph2 相互作用，并且它们与 Dph3 形成复合物。蛋白-蛋白相互作用图谱显示，Dph1-Dph3 复合物的形成可以通过逐步的 DPH1 基因截断来进行剖析。这确定了 Dph1 的 N 端和 C 端结构域对于二氢喋呤的合成、DT 作用以及另一种微生物 eEF2 抑制剂 sordarin 的细胞毒性至关重要。有趣的是，dph1 截断突变体对 DPH5 的过表达敏感，DPH5 是从 Dph1-Dph3 产生的第一个二氢喋呤途径中间产物合成二氢喋呤所必需的基因。这与 dph6 突变体形成鲜明对比，dph6 突变体也缺乏形成二氢喋呤的能力，但对过量 Dph5 水平的生长抑制具有抗性。从定点突变来看，酰胺化反应本身依赖于 Dph6 中保守的 ATP 结合结构域，当该结构域发生改变时，会阻止二氢喋呤的形成，并赋予 sordarin 对 eEF2 抑制的抗性。
