Self-activating G protein α subunits engage seven-transmembrane regulator of G protein signaling (RGS) proteins and a Rho guanine nucleotide exchange factor effector in the amoeba Naegleria fowleri

• Published in: The Journal of biological chemistry Vol. 298; no. 8; p. 102167
• Main Authors: Bosch, Dustin E., Jeck, William R., Siderovski, David P.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.08.2022; American Society for Biochemistry and Molecular Biology
• Subjects: brain; cell signaling; enzyme kinetics; enzyme structure; G protein; GTPase activating protein; microbiology; protein crystallization; protein structure; signal transduction; signal transduction; PLC; microbiology; GEF; brain; enzyme kinetics; enzyme structure; protein structure; P-loop; protein crystallization; SPR; cell signaling; GAP; r.m.s.d; GPCR; AMF; MSA; G protein; GTPase activating protein; RGS
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2022.102167

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 Gα12/13 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.