Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor

Abstract

Resistance to inhibitors of cholinesterase 8A (Ric8A) is an essential regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor and a chaperone. We report two crystal structures of Ric8A, one in the apo form and the other in complex with a tagged C-terminal fragment of Gα. These structures reveal two principal domains of Ric8A: an armadillo-fold core and a flexible C-terminal tail. Additionally, they show that the Gα C-terminus binds to a highly-conserved patch on the concave surface of the Ric8A armadillo-domain, with selectivity determinants residing in the Gα sequence. Biochemical analysis shows that the Ric8A C-terminal tail is critical for its stability and function. A model of the Ric8A/Gα complex derived from crosslinking mass spectrometry and molecular dynamics simulations suggests that the Ric8A C-terminal tail helps organize the GTP-binding site of Gα. This study lays the groundwork for understanding Ric8A function at the molecular level.

Ric‐8A, a GEF, and a Chaperone for G Protein α‐Subunits: Evidence for the Two‐Faced Interface

“… Not only could the “open” conformation of Ric‐8A accommodate Gα; it was also consistent with the SAXS profile of Ric‐8A1‐452 (Figure 4C). Parsimonious models of Ric‐8A1‐452 and Ric‐8A1‐492 in complex with minimal Gα (miniGα) and full‐length Gα have been proposed based on the GPCR‐bound conformations of Gα and an assumed “open” conformation of Ric‐8A . However, an attempt to validate these models using SAXS analysis of the Ric‐8A1‐492/miniGα i complex revealed strong disagreement between the theoretical and experimental SAXS profiles .…”

“…For a long period of time, Ric‐8A escaped attempts to determine its structure by means of X‐ray crystallography. A probable reason for the “reluctance” of Ric‐8 to form well‐diffracting crystals became apparent following the recent solution of its structure . The structure of the active nearly full‐length Ric‐8A1‐492 revealed two principal domains of Ric‐8A: an armadillo‐like core (residues about 1–426) and an unstructured C‐terminal tail (residues 427–492) .…”

“…Residues Gα t 335‐346 form an α‐helix, whereas Gα t 347‐350 is in an extended conformation. Mutational analysis of the interface residues from both Gα and Ric‐8A confirmed that the same interface is utilized for the Ric‐8A interaction with the full‐length Gα . Remarkably, the side of Gα t α5‐helix that interacts with Ric‐8A is similar to that for the interaction with GPCRs in the fully coupled nucleotide‐free state (Figure 1C,E,F).…”

“…The higher B‐factors for the N‐ and C‐terminal portions of Ric‐8A1‐423 indicate higher conformational flexibility. B) Overlay of the apo Ric‐8A structure (green) and the “open” conformation of the Ric‐8A core domain (orange), which resulted from the SMD simulation with force applied to the Ric‐8A region that clashed with Gα . The “open” conformation of Ric‐8A would allow the avoidance of clashes with Gα in a Ric‐8A/Gα complex modeled using Gα in a GPCR‐bound conformation.…”

“…Indeed, the distal C‐tail is important for the GEF activity of Ric‐8A. Various degrees of impairment of the GEF activity have been reported for the truncated Ric‐8A1‐452 construct, but a preponderance of the data suggests that it is a very poor GEF compared to the full‐length Ric‐8A or Ric‐8A1‐492 . Moreover, a potential Gα‐binding site has been proposed for residues Ric‐8A455‐470 .…”