Cytotoxic necrotizing factors (CNFs) are bacterial single‐chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three‐dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full‐length Yersinia pseudotuberculosis CNF_Y. CNF_Y consists of five domains (D1–D5), and by integrating structural and functional data, we demonstrate that D1–3 act as export and translocation module for the catalytic unit (D4–5) and for a fused β‐lactamase reporter protein. We further found that D4, which possesses structural similarity to ADP‐ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4–5 fragment. This liberates D5 from a semi‐blocked conformation in full‐length CNF_Y, leading to higher deamidation activity. Finally, we identify CNF translocation modules in several uncharacterized fusion proteins, which suggests their usability as a broad‐specificity protein delivery tool.

中文翻译：

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细菌细胞毒性坏死因子 CNFY 的晶体结构揭示了中毒的分子构件

细胞毒性坏死因子 (CNF) 是细菌单链外毒素，通过激活宿主细胞 Rho GTP 酶来调节细胞因子/致癌和炎症过程。为了实现这一点，它们被分泌出来，结合表面受体以诱导内吞作用，并将催化单位转移到细胞质中，使宿主细胞中毒。仍然缺乏可以深入了解潜在机制的三维结构。在这里，我们确定了全长假结核耶尔森氏菌CNF _Y的晶体结构。CNF _Y由五个结构域 (D1-D5) 组成，通过整合结构和功能数据，我们证明 D1-3 作为催化单元 (D4-5) 和融合 β-内酰胺酶报告蛋白的输出和易位模块。我们进一步发现 D4 与 ADP-核糖基转移酶具有结构相似性，但没有等效的催化活性，在游离 D4-5 片段的晶体结构中改变了与 D5 广泛相互作用的位置。这将 D5 从全长 CNF _Y的半封闭构象中释放出来，导致更高的脱酰胺活性。最后，我们在几种未表征的融合蛋白中鉴定了 CNF 易位模块，这表明它们可用作广泛的特异性蛋白质递送工具。