Srivastava_2019_J.Biol.Chem_294_17875

Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein-coupled receptor (GPCR)-independent regulator of G protein alpha-subunits (Galpha), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and Galpha hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent Galpha proteins. Here, we examined the structure of the complex of Ric8A with minimized Galphai (miniGalphai) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGalphai complex by steered molecular dynamics (SMD) simulations. A small set of models of the complex featured minimal clash scores, excellent agreement with the experimental SAXS data, and a large-scale rearrangement of the signal-transducing alpha5-helix of Galpha away from its beta-sheet core. The resulting interface involved the Galpha alpha5-helix bound to the concave surface of Ric8A and the Galpha beta-sheet that wraps around the C-terminal part of the Ric8A armadillo domain, leading to a severe disruption of the GDP-binding site. Further modeling of the flexible C-terminal tail of Ric8A indicated that it interacts with the effector surface of Galpha. This smaller interface may enable the Ric8A-bound Galpha to interact with GTP. The two-interface interaction with Galpha described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.