Abstract
Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein-coupled receptor (GPCR)-independent regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and Gα hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent Gα proteins. Here, we examined the structure of the complex of Ric8A with minimized Gαi (miniGαi) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGαi 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 α5-helix of Gα away from its β-sheet core. The resulting interface involved the Gα α5-helix bound to the concave surface of Ric8A and the Gα β-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 Gα. This smaller interface may enable the Ric8A-bound Gα to interact with GTP. The two-interface interaction with Gα described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.
Highlights
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
We evaluated the theoretical small-angle X-ray scattering (SAXS) profile of the Ric8A1– 452/⌬␣NminiG␣i model lacking residues 453– 492 of Ric8A, which served as a template for models 1 and 2 (Fig. 2A)
The analysis of the structure of the Ric8A1– 492/⌬␣NminiG␣i complex in solution by SAXS reported in this study led us to propose a new model of this complex
Summary
Resistance to inhibitors of cholinesterase 8; GEF, guanine nucleotide exchange factor; GPCR, G protein– coupled receptor; miniG␣i, minimized G␣i; RD, Ras-like domain; SMD, steered molecular dynamics; SAXS, small-angle X-ray scattering; HD, helical domain; TCEP, tris(2-carboxyethyl)phosphine; SEC, size-exclusion chromatography. Activation by Ric8A have been provided by the recent crystal structure of the complex of Ric8A with the C-terminal fragment of G␣ corresponding to the ␣5-helix [10]. Based on this structure, we modeled the complex of Ric8A with miniG␣i and the full-length G␣i subunit [10]. The resulting models are in excellent agreement with the experimental SAXS profile, and they feature large rearrangement of the G␣ ␣5-helix
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