The free-living amoeba Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis and is highly resistant to current therapies, resulting in mortality rates >97%. As many therapeutics target G protein–centered signal transduction pathways, further understanding the functional significance of G protein signaling within N. fowleri should aid future drug discovery against this pathogen. Here, we report that the N. fowleri genome encodes numerous transcribed G protein signaling components, including G protein–coupled receptors, heterotrimeric G protein subunits, regulator of G protein signaling (RGS) proteins, and candidate Gα effector proteins. We found N. fowleri Gα subunits have diverse nucleotide cycling kinetics; Nf Gα5 and Gα7 exhibit more rapid nucleotide exchange than GTP hydrolysis (i.e., “self-activating” behavior). A crystal structure of Nf Gα7 highlights the stability of its nucleotide-free state, consistent with its rapid nucleotide exchange. Variations in the phosphate binding loop also contribute to nucleotide cycling differences among Gα subunits. Similar to plant G protein signaling pathways, N. fowleri Gα subunits selectively engage members of a large seven-transmembrane RGS protein family, resulting in acceleration of GTP hydrolysis. We show Nf Gα2 and Gα3 directly interact with a candidate Gα effector protein, RGS-RhoGEF, similar to mammalian Gα_12/13 signaling pathways. We demonstrate Nf Gα2 and Gα3 each engage RGS-RhoGEF through a canonical Gα/RGS domain interface, suggesting a shared evolutionary origin with G protein signaling in the enteric pathogen Entamoeba histolytica. These findings further illuminate the evolution of G protein signaling and identify potential targets of pharmacological manipulation in N. fowleri.

自由生活的阿米巴Naegleria fowleri是原发性阿米巴脑膜脑炎的病原体，对目前的治疗具有高度耐药性，导致死亡率 >97%。由于许多治疗剂针对以 G 蛋白为中心的信号转导通路，因此进一步了解福氏猪瘟内 G 蛋白信号传导的功能意义应​​该有助于未来发现针对这种病原体的药物。在这里，我们报告N. fowleri基因组编码许多转录的 G 蛋白信号成分，包括 G 蛋白偶联受体、异源三聚体 G 蛋白亚基、G 蛋白信号 (RGS) 蛋白调节因子和候选 Gα 效应蛋白。我们发现了N. fowleriGα亚基具有不同的核苷酸循环动力学；Nf Gα5 和 Gα7 表现出比 GTP 水解更快的核苷酸交换（即“自激活”行为）。Nf Gα7 的晶体结构突出了其无核苷酸状态的稳定性，与其快速的核苷酸交换一致。磷酸结合环的变化也有助于 Gα 亚基之间的核苷酸循环差异。与植物 G 蛋白信号通路类似，福氏猪笼草Gα 亚基选择性地参与大型七跨膜 RGS 蛋白家族的成员，从而加速 GTP 水解。我们展示了 Nf Gα2 和 Gα3 直接与候选 Gα 效应蛋白 RGS-RhoGEF 相互作用，类似于哺乳动物 Gα _12/13信号通路。我们证明 Nf Gα2 和 Gα3 均通过规范的 Gα/RGS 结构域界面与 RGS-RhoGEF 结合，这表明肠道病原体溶组织内阿米巴中的 G 蛋白信号传导具有共同的进化起源。这些发现进一步阐明了 G 蛋白信号传导的演变，并确定了福氏猪瘟中药理学操作的潜在目标。