Large-scale conformational rearrangement of the 5-helix of G subunits in complex with the guanine nucleotide exchange factor Ric8A

Abstract

Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein- coupled receptor (GPCR)-independent regulator of G protein -subunits (G), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and G hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent G proteins. Here, we examined the structure of the complex of Ric8A with minimized Gi (miniGi) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGi 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 5-helix of G away from its β-sheet core. The resulting interface involved the G 5-helix bound to the concave surface of Ric8A and the G-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 G. This smaller interface may enable the Ric8A-bound G to interact with GTP. The two-interface interaction with G described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.