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Structural Underpinnings of Ric8a Function As a G-Protein О ARTICLE https://doi.org/10.1038/s41467-019-11088-x OPEN Structural underpinnings of Ric8A function as a G-protein α-subunit chaperone and guanine-nucleotide exchange factor Dhiraj Srivastava1, Lokesh Gakhar 2,3 & Nikolai O. Artemyev 1,4 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 1234567890():,; 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. 1 Department of Molecular Physiology and Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. 2 Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. 3 Protein Crystallography Facility, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. 4 Department of Ophthalmology and Visual Sciences, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA. Correspondence and requests for materials should be addressed to N.O.A. (email: [email protected]) NATURE COMMUNICATIONS | (2019) 10:3084 | https://doi.org/10.1038/s41467-019-11088-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-11088-x protein-coupled receptors (GPCRs) and their cognate It had been proposed that Ric8 might interact with the con- heterotrimeric G proteins (Gαβγ) are the key components formationally sensitive switch II region of Gα, which is occluded G βγ8,23,24 of a major cellular signaling pathway that is activated in the heterotrimer by G . This interaction may also when agonist binds to the GPCR, enabling its interaction with explain the selectivity of Ric8 for the GDP-bound state of Gα8. Gαβγ and catalyzing the release of GDP from Gα. This enables Notwithstanding these differences, notable parallels between G Gα to bind GTP, triggering dissociation of the signaling species protein activation by Ric8A and GPCRs were suggested by some GαGTP and Gβγ, which modulate numerous downstream effec- of the available biochemical data. Like GPCRs, Ric8A has been tors. Thus, GPCRs serve as guanine nucleotide-exchange factors shown to induce separation of RD and HD of Gα, and to do so by (GEFs) for G proteins. However, G proteins can also be activated perturbing the secondary structure surrounding the guanine- by nonreceptor GEFs. Notable among these are the resistance to binding site within the RD25. A second parallel is that the key inhibitors of cholinesterase 8 (Ric8) proteins, which regulate G- Ric8A interaction site of Gα has been localized to the C-terminal protein biology in species ranging from slime molds to verte- α5 helix26. brates and, in contrast to GPCRs, act on monomeric Gα sub- To gain mechanistic insights into the function of Ric8 and its units1–5. Whereas the genomes of invertebrates encode a single interaction with Gα, we solve the crystal structures of both apo ancestral Ric8 isoform that is capable of interacting with Gα Ric8A and Ric8A in complex with the C-terminal fragment of 6,7 α α subunits of all types , their vertebrate counterparts encode two transducin- (G t) attached to a maltose-binding protein (MBP) isoforms, each of which regulates a particular subset of Gα sub- tag. These structures reveal that Ric8A has two major modules: α α α α 8–11 units: Ric8A (G i/t,G q, and G 12/13) and Ric8B (G s) . The the core armadillo (ARM)-repeat domain, composed of 8 ARM interaction of Ric8 with GDP-bound Gα stimulates release of repeats, and a flexible C-terminal tail. In addition, they show that GDP, leading to the formation of a stable intermediate complex the concave surface of the Ric8A armadillo domain encompasses of Ric8 and nucleotide-free Gα. Once Gα binds GTP it dissociates a conserved binding site for the C-terminus of Gα. Our bio- from Ric8, and thus the nucleotide-exchange cycle on Gα is chemical studies demonstrate that the C-terminal tail is impor- completed8,12. tant for the stability of the Ric8A protein overall, and key for its Although the GEF activity of Ric8A and Ric8B was initially GEF function. Modeling of the Ric8A/Gα complex and flexible thought to account for the ability of these proteins to positively C-terminal tail of Ric8A provides a foundation for understanding regulate G-protein signaling, mounting evidence suggests that the the molecular mechanisms that underlie the stability, GEF Ric8 proteins can also serve as ubiquitous chaperones for Gα- function, and chaperone function of Ric8A. subunits13–15. The fact that the chaperone function of Ric8 augments G-protein signaling could readily explain many, if not all, biological effects of the protein. Although it remains unclear Results whether the GEF or chaperone function of Ric8 proteins is pre- Ric8A binds the C-terminus of Gα with high affinity. The C- dominant with respect to its biological effects, we reasoned that terminal 18-mer peptide of Gαi corresponding to Gαt333–350, the structure of the Ric8/Gα complex might hold the key to was previously shown to bind to Ric8A26. To identify C-terminal understanding both. This complex underlies the GEF activity of constructs of Gα that are suitable for crystallization with Ric8A, α Ric8 and may, in fact, be similar to the folding intermediate that we tested one in which the 11 C-terminal residues of G t were is present during the biosynthesis of Gα12. fused to the B1 domain of Streptococcal protein G (GB1- α – The mechanism underlying the ability of GPCRs to function as G t340 350) and a second in which the 24 C-terminal residues of GEFs has been extensively investigated. Structures of GPCR/Gαβγ Gαt were fused to MBP (MBP-Gαt327–350). For Ric8A, we used a complexes have revealed, in atomic detail, GPCR-induced struc- construct that is truncated at the C-terminus (Ric8A1–492) and tural perturbations of Gα that lead to the release of GDP16–19. that had been reported to retain the full functionality of Ric8A26. Gα subunits feature two key domains, the Ras-like domain (RD) Elution profiles generated by size-exclusion chromatography and the α-helical domain (HD)20. The RD binds the nucleotide (SEC) indicated that both fusion proteins formed complexes with and interacts with the HD, with the latter serving as a lid over the Ric8A1–492 at nearly 1:1 stoichiometry (Supplementary Fig. 1), nucleotide binding site. Agonist-bound GPCR engages Gαβγ at suggesting that the 11 C-terminal residues of Gαt are sufficient for α α fi two key sites of G : the C-terminal 5 helix and the N-terminal a high-af nity (submicromolar Kd) interaction. αN-β1 loop. The largest GPCR-induced conformational change in Next, we evaluated the effects that binding of the 11-mer and α α α α – G is an outward translation with rotation of the 5 helix that 18-mer C-terminal peptides of G t (G t340 350 and β α α – leads to a displacement of the guanine ring binding loop 6- 5of G t333 350, respectively) had on the thermal stability of Gα16. Changes associated with the interaction between the Ric8A1–492. To this end, we used differential scanning receptor and the αN-β1 loop propagate to and disturb the fluorimetry (DSF) (Fig. 1a). These tests revealed that β α α – α – phosphate-binding P-loop ( 1- 1). The dual disruption of G t333 350, but not G t340 350, had a strong stabilization the guanine-nucleotide binding site is accompanied by weakening effect (~9 °C shift) on Ric8A1–492 (Fig.1a). We infer from the of the RD/HD interface and stabilization of conformational states protein stabilization effect that the longer peptide more fully α in which RD and HD are dynamically separated, facilitating the recapitulates the C-terminal interaction of G t with Ric8A. The 16,21 fi α – – escape of GDP . kinetics and af nity of binding for G t333 350 and Ric8A1 492 In contrast to the wealth of structural information available were quantitated using biolayer interferometry (BLI), with N- α – regarding the interactions between GPCRs and G proteins, data at terminally biotinylated G t333 350 attached to a streptavidin atomic level have not been available for Ric8 proteins, either alone biosensor. The binding and dissociation kinetics for the α – α – or in complex with G . A lack of sequence similarity between Ric8A1 492/G t333 350 interaction were consistent with a 1:1 Ric8 isoforms and other proteins has precluded homology binding model, with the average association constant (ka) modeling of its structure. However, protein-fold recognition measured at 1.3 ± 0.3 × 105 M−1s−1 (mean ± SD) and the average −1 = algorithms have predicted that Ric8 adopts an armadillo-like dissociation constant (kd) at 0.031 ± 0.008 s , yielding a KD 22 fold . The complete lack of structural relatedness between kd/ka of 0.24 µM (Fig.
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