The Ras–extracellular signal–regulated kinase pathway is critical for controlling cell proliferation, and its aberrant activation drives the growth of various cancers. Because many pathogens produce toxins that inhibit Ras activity, efforts to develop effective Ras inhibitors to treat cancer could be informed by studies of Ras inhibition by pathogens. Vibrio vulnificus causes fatal infections in a manner that depends on multifunctional autoprocessing repeats-in-toxin, a toxin that releases bacterial effector domains into host cells. One such domain is the Ras/Rap1-specific endopeptidase (RRSP), which site-specifically cleaves the Switch I domain of the small GTPases Ras and Rap1. We solved the crystal structure of RRSP and found that its backbone shares a structural fold with the EreA/ChaN-like superfamily of enzymes. Unlike other proteases in this family, RRSP is not a metalloprotease. Through nuclear magnetic resonance analysis and nucleotide exchange assays, we determined that the processing of KRAS by RRSP did not release any fragments or cause KRAS to dissociate from its bound nucleotide but instead only locally affected its structure. However, this structural alteration of KRAS was sufficient to disable guanine nucleotide exchange factor–mediated nucleotide exchange and prevent KRAS from binding to RAF. Thus, RRSP is a bacterial effector that represents a previously unrecognized class of protease that disconnects Ras from its signaling network while inducing limited structural disturbance in its target.

Ras 细胞外信号调节激酶通路对于控制细胞增殖至关重要，其异常激活会驱动各种癌症的生长。由于许多病原体会产生抑制 Ras 活性的毒素，因此可以通过研究病原体对 Ras 的抑制作用来开发有效的 Ras 抑制剂来治疗癌症。创伤弧菌导致致命感染的方式取决于毒素中的多功能自动处理重复序列，这是一种将细菌效应结构域释放到宿主细胞中的毒素。一个这样的结构域是 Ras/Rap1 特异性内肽酶 (RRSP)，它位点特异性地切割小 GTPases Ras 和 Rap1 的 Switch I 结构域。我们解决了 RRSP 的晶体结构，发现它的骨架与 EreA/ChaN 样酶超家族有一个结构折叠。与该家族中的其他蛋白酶不同，RRSP 不是金属蛋白酶。通过核磁共振分析和核苷酸交换分析，我们确定 RRSP 对 KRAS 的处理不会释放任何片段或导致 KRAS 与其结合的核苷酸解离，而只是局部影响其结构。然而，KRAS 的这种结构改变足以禁用鸟嘌呤核苷酸交换因子介导的核苷酸交换并阻止 KRAS 与 RAF 结合。因此，RRSP 是一种细菌效应物，它代表了一种以前未被识别的蛋白酶类，它将 Ras 与其信号网络断开，同时在其靶标中诱导有限的结构干扰。