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

Abstract

Resistance to inhibitors of cholinesterase 8A (Ric‐8A) is a prominent non‐receptor GEF and a chaperone of G protein α‐subunits (Gα). Recent studies shed light on the structure of Ric‐8A, providing insights into the mechanisms underlying its interaction with Gα. Ric‐8A is composed of a core armadillo‐like domain and a flexible C‐terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C‐terminus appears to mediate formation of the initial Ric‐8A/GαGDP intermediate, followed by the formation of a stable nucleotide‐free complex. The latter event involves a large‐scale dislocation of the Gα α5‐helix that produces an extensive primary interface and disrupts the nucleotide‐binding site of Gα. The distal portion of the C‐terminal tail of Ric‐8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two‐site Gα interface of Ric‐8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric‐8A.

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

“…The G protein signaling cycle is regulated by several proteins, including GEFs (e.g., GPCRs, GIV/Girdin, or Ric-8A) and GTPase-activating proteins [GAPs; e.g., regulators of G protein signaling (RGS)]. 48 , 49 , 50 , 51 , 52 As our data support slow AHD closing enabled by the long-lived intermediate state of Gαs after GTPγS binding, we searched for an AHD-binding protein that might regulate the AHD closing kinetics. To this end, we employed Y2H library screening with the Gαs AHD as bait and identified melanoma-associated antigen D2 (MAGE D2, NM_014599) as a novel Gαs AHD-binding protein ( Figure S8 A).…”

Gαs slow conformational transition upon GTP binding and a novel Gαs regulator

“…The G-protein signaling cycle is regulated by several proteins, including GEFs (e.g., GPCRs, GIV/Girdin, or Ric-8A) and GTPase-activating proteins [GAPs; e.g., regulators of G protein signaling (RGS)] (Kach et al, 2012; Kalogriopoulos et al, 2019; Siderovski and Willard, 2005; Srivastava and Artemyev, 2020; Srivastava et al, 2019). As our data support slow AHD closing enabled by the long-lived intermediate state of Gαs after GTPγS binding, we searched for an AHD-binding protein that might regulate the AHD closing kinetics.…”

Transducin Partners Outside the Phototransduction Pathway

“…Depending on the Ric-8A function in a given system, the structures can represent either the GEF complex intermediate with an empty-pocket for nucleotide binding or the folding intermediate of Gα during its biosynthesis. The Ric-8A/Gα complexes reveal two remarkable features: a large displacement of the α5-helix of Gα from the β-sheet cradle of the Ras-like domain, and a unique interaction of the C-terminal helix of Ric-8A with the switch II/α3-helix region of Gα ( Srivastava and Artemyev, 2019 , 2020 ; McClelland et al, 2020 ; Seven et al, 2020 ). The interaction of Ric-8A with the C-terminus/α5-helix of Gα is central to both the GEF and the chaperone activity.…”

“…When Ric-8A acts as a chaperone, this interaction induces the folding of the α5-helix outside the β-sheet cradle of partially folded Gα ( Srivastava et al, 2019 ; McClelland et al, 2020 ). The interaction of the C-terminal helix of Ric-8A with the switch II/α3-helix region of Gα likely promotes GTP-binding to Gα thereby concluding either the GEF or the chaperone cycle of Ric-8A ( Srivastava and Artemyev, 2019 , 2020 ; McClelland et al, 2020 ; Seven et al, 2020 ). We demonstrated that Ric-8A is expressed throughout the retina.…”

“…Upon binding of GTP, Gα is released from Ric-8A and the α5-helix replaces Ric-8A in stabilizing the β-sheet core of Gα. The GTP-binding site in the Ric-8A/Gα complex is disorganized to a greater extent compared to that in the GPCR/Gαβγ complexes ( McClelland et al, 2020 ; Seven et al, 2020 ; Srivastava and Artemyev, 2020 ). Remarkably, the distal portion of the C-terminal tail of Ric-8A forms a unique smaller secondary interface with the switch II/α3-helix region of Gα, which appears to assist GTP-binding ( Srivastava and Artemyev, 2019 ; McClelland et al, 2020 ; Seven et al, 2020 ; Figure 1C ).…”