Structural Determinants of RGS-RhoGEF Signaling Critical to Entamoeba histolytica Pathogenesis

G protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion. Coexpression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the Dbl homology domain Rho-binding surface and the pleckstrin homology domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF interface confirms a canonical “regulator of G protein signaling” domain rather than a RhoGEF-RGS (“rgRGS”) domain, suggesting a convergent evolution toward heterotrimeric and small G protein cross-talk. [Display omitted] ▸ E. histolytica expresses a convergently evolved RGS-RhoGEF protein ▸ EhRGS-RhoGEF possesses a conventional nine-helix RGS domain ▸ Activated EhRGS-RhoGEF induces S2 cell spreading through Rac1/2 ▸ EhRGS-RhoGEF modulates amoebic migration and host cell killing Intestinal parasite Entamoeba histolytica has components of a heterotrimeric G-protein signaling pathway. Here, Bosch et al. identify EhRGS-RhoGEF as a G-protein effector that modulates multiple pathogenic processes. Structural analysis provides insight into the autoinhibitory mechanism and evolutionary origin.