Protonation of key residues in the diphtheria toxin translocation (T)-domain triggered by endosomal acidification is critical for inducing a series of conformational transitions critical for the cellular entry of the toxin. Previous experiments revealed the importance of histidine residues in modulating pH-dependent transitions. They suggested the presence of a “safety latch” preventing premature refolding of the T-domain by a yet poorly understood mechanism. Here, we used constant-pH molecular dynamics simulations to systematically investigate the protonation sequence in the wild-type T-domain and the following mutants: H223Q, H257Q, E259Q, and H223Q/H257Q. Comparison of these computational results with previous experimental data on T-domain stability and activity with the H-to-Q replacements confirms the role of H223 (pKa = 6.5) in delaying the protonation of the main trigger, H257 (pKa = 2.2 in the WT and pKa = 4.9 in H223Q). Our calculations also reveal a very low pKa for a neighboring acidic residue E259, which does not get protonated even during simulations at pH 3. This residue also contributes to the formation of the safety latch, with the pKa of H257 increasing from 2.2 to 5.1 upon E259Q replacement. In contrast, the latter replacement has virtually no effect on the protonation of the H223. Thus, we conclude that the interplay of the protonation in the H223/H257/E259 triad has evolved to prevent triggering the accidental refolding of the T-domain by a fluctuation in the protonation of the main trigger at neutral pH, before the incorporation of the toxin inside the endosome. Subsequent acidification of the endosome overcomes the safety latch and triggers conformational switching via repulsion of H223+ and H257+. This protonation/conformation relationship corroborates experimental findings and offers a detailed stepwise molecular description of the transition mechanism, which can be instrumental in optimizing the potential applications of the T-domain for targeted delivery of therapies to tumors and other diseased acidic tissues.

中文翻译：

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白喉毒素易位域中质子化触发的构象转换的恒定 pH MD 模拟


由内体酸化触发的白喉毒素易位 （T） 结构域中关键残基的质子化对于诱导一系列对毒素细胞进入至关重要的构象转换至关重要。以前的实验揭示了组氨酸残基在调节 pH 依赖性转变中的重要性。他们提出了一个“安全闩锁”的存在，通过一种尚不为人知的机制来防止 T 结构域过早重新折叠。在这里，我们使用恒定 pH 分子动力学模拟系统地研究了野生型 T 结构域和以下突变体中的质子化序列：H223Q、H257Q、E259Q 和 H223Q/H257Q。将这些计算结果与先前关于 T 域稳定性和活性与 H-to-Q 替代品的实验数据进行比较，证实了 H223 （pKa = 6.5） 在延迟主要触发因素 H257 的质子化中的作用（WT 中的 pKa = 2.2，H223Q 中的 pKa = 4.9）。我们的计算还揭示了相邻酸性残基 E259 的 pKa 非常低，即使在 pH 值为 3 的模拟过程中，该残基也不会被质子化。该残基还有助于安全锁的形成，更换 E259Q 后，H257 的 pKa 从 2.2 增加到 5.1。相比之下，后者的取代对 H223 的质子化几乎没有影响。因此，我们得出结论，H223/H257/E259 三联体中质子化的相互作用已经进化为防止在中性 pH 下通过主要触发物质子化的波动触发 T 结构域的意外重折叠，在毒素掺入内体之前。随后的内体酸化克服了安全锁，并通过排斥 H223 + 和 H257 + 触发构象转换。 这种质子化/构象关系证实了实验结果，并提供了过渡机制的详细逐步分子描述，这有助于优化 T 结构域的潜在应用，以便将治疗靶向递送到肿瘤和其他患病的酸性组织。