Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor

Abstract

Cholecystokinin A receptor (CCK_AR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCK_AR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including G_s, G_i and G_q. However, the basis for G-protein coupling promiscuity and ligand recognition by CCK_AR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCK_AR in complex with G_s, G_i and G_q heterotrimers, respectively. CCK_AR presents a similar conformation in the three structures, whereas conformational differences in the ‘wavy hook’ of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCK_AR and uncover the mechanism of receptor recognition by sulfated CCK-8.

Cryo-EM structures of sulfated cholecystokinin 8 bound to the cholecystokinin A receptor in complex with G_s, G_i and G_q heterotrimers reveal structural determinants for G-protein coupling selectivity.

Main

Cholecystokinin (CCK), one of the earliest discovered gastrointestinal hormones, participates in gallbladder contraction and pancreatic enzyme secretion. It also acts as a neurotransmitter and is extensively distributed throughout the nervous system¹. Selective cleavage of the CCK precursor produces a series of bioactive isoforms of different lengths, with CCK-58, CCK-33, CCK-22 and CCK-8 comprising the major peptide fragments in humans. However, the carboxy-terminal octapeptide CCK-8 (DYMGWMDF) is well conserved across species and is the smallest form that retains the full range of biological actions², mediated by two CCK receptor subtypes (CCK_AR and CCK_BR), which are present throughout the central nervous system and the gut. CCK_AR is primarily expressed in the alimentary tract, while CCK_BR is mainly found in the brain and the stomach³. CCK_AR has an ~500-fold higher affinity to CCK with a sulfated tyrosine, whereas CCK_BR discriminates poorly between sulfated and non-sulfated CCK⁴.

CCK regulates appetite and food intake primarily through CCK_AR on the vagal afferent neurons^5–8, making CCK_AR an attractive therapeutic target for obesity. However, the development of drugs against CCK_AR is challenging, partly due to limited efficacy and safety concerns. Although several drug candidates are undergoing clinical trials, none has been approved so far^9,10. Extensive efforts have been made to elucidate the mechanism of agonism at CCK_AR through mutagenesis studies based on modeled receptor structures^11–15, but the lack of precise structural information largely impedes our understanding of the molecular details of ligand recognition and receptor activation, and thus the drug discovery targeting CCK_AR.

Most G-protein-coupled receptors (GPCRs) are known to couple with a specific subtype of G protein to elicit intracellular signal transduction^16–21. There are four G-protein subtypes—stimulatory G protein (G_s), inhibitory G proteins (G_i), G_q and G_12/13—participating in signaling pathways involving cyclic adenosine monophosphate (G_s and G_i), calcium (G_q) and small G protein (G_12/13). A number of GPCR–G protein complex structures reported recently reveal that the primary determinants of G-protein coupling selectivity reside in the C-terminal α5-helix of the Gα subunit and relative outward movement of transmembrane helix 6 (TM6)^22,23. However, CCK_AR is different from most GPCRs as a result of its ability to couple with several G-protein subtypes. Activation of CCK_AR elicits a diversified G-protein coupling pattern²⁴, predominantly G_q (ref. ²⁵), but G_s (ref. ²⁶), G_i (refs. ^25,27) and G₁₃ (refs. ^28,29) all play roles in CCK_AR signaling. This unique feature makes CCK_AR an ideal model to study G-protein selectivity and promiscuity (Fig. 1a). Here, we report three cryo-EM structures of sulfated CCK-8-activated CCK_AR in complex with heterotrimeric G_q, G_s or G_i protein, respectively. These structures reveal the unique binding mode in ligand recognition and the structural determinants responsible for the G-protein selectivity and promiscuity of CCK_AR.

Fig. 1. Cryo-EM structures of CCK_AR–G protein complexes.

a, Schematic of G-protein coupling promiscuity of CCK_AR. b–g, Three-dimensional maps and models of the CCK-8–CCK_AR–G_q–scFv16 (b,c), CCK-8–CCK_AR–G_s (d,e) and CCK-8–CCK_AR–G_i–scFv16 (f,g) complexes. CCK-8, magenta; G_q-coupled CCK_AR, green (b,c); G_s-coupled CCK_AR, pink (d,e); G_i-coupled CCK_AR, dark yellow (f,g); Gα_q, orange; Gα_s, blue; Gα_i, cyan; Gβ, light blue; Gγ, yellow; scFv16, light purple.

Overall structures of CCK_AR coupled to different G proteins

The structures of sulfated CCK-8-bound CCK_AR in complex with G_q, G_s or G_i heterotrimers were determined by single-particle cryo-EM at global resolutions of 2.9 Å, 3.1 Å and 3.2 Å, respectively (Fig. 1b–g, Supplementary Figs. 1–3 and Supplementary Table 1). Sulfated CCK-8 (DY^SO3HMGMWDF-NH₂), the highest-affinity natural ligand of CCK_AR⁴, was used to assemble the CCK_AR–G protein complexes. Three G-protein subtypes were engineered to stabilize the CCK_AR–G protein complexes (Supplementary Fig. 4). Gα_q is chimerized by replacing its αN-helix with the equivalent region of Gα_i1 to facilitate scFv16 binding³⁰. Gα_s was modified based on the mini-Gα_s that was used in the crystal structure determination of the G_s-coupled adenosine A_2A receptor (A_2AR)³¹. Two dominant-negative (DN) mutations (G203A and A326S³²) were introduced to Gα_i1, and corresponding DN mutations at equivalent sites of Gα_s and Gα_q were also introduced (Supplementary Fig. 4b). Unless otherwise specified, G_q, G_s and G_i refer to the respective engineered G proteins that are used in the CCK_AR structure determination.

The final structures of the CCK-8–CCK_AR–G protein complexes contain sulfated CCK-8 (residues D^1P-F^8P), Gα Ras-like domain, Gβγ subunits, scFv16 and the CCK_AR residues (E38^N_term-F385^8.58, superscripts refer to Ballesteros–Weinstein numbering³³). The majority of amino-acid side chains, including CCK-8, transmembrane domain (TMD), intracellular loops (ICLs 1–3) and extracellular loops (ECLs 1–3) were well resolved in the final models (Supplementary Fig. 5). Thus, the complex structures provide reliable details to study mechanisms of ligand recognition and G-protein coupling.

Globally, CCK_AR adopts similar overall conformations in all three structures, with an all-atom root-mean-square deviation (r.m.s.d.) of 0.84 for G_q/G_s-coupled receptors and 1.03 for G_q/G_i-coupled receptors. The structure of the CCK-8–CCK_AR–G_q complex, which has the highest resolution at 2.9 Å, was used for detailed analysis and mechanistic evaluation of ligand recognition and receptor activation. The inactive and active structures of the closed homolog receptors (inactive: ghrelin receptor, PDB 6KO5³⁴; active: neurotensin receptor 1 (NTSR1), PDB 6OS9¹⁸), all belonging to the β-branch of the rhodopsin family, are applied for structural comparison. CCK_AR presents a fully active conformation, resembling the G_i-coupled NTSR1, displaying an ~9-Å outward movement of TM6 (measured at the Cα of the residue at position 6.27 in CCK_AR and the ghrelin receptor) and an ~4-Å inward shift of TM7 (Cα carbons of Y7.53) compared with the inactive ghrelin receptor (Extended Data Fig. 1a,b). Similar to the active NTSR1 complex, the conserved residues in the ‘micro-switches’ (PIF, ERY, CWxP and NPxxY) of CCK_AR display the conserved conformations observed in active GPCRs (Extended Data Fig. 1c).

Extended Data Fig. 1. Active conformation of CCK_AR.

a-b, Structural comparison of inactive ghrelin receptor (grey), active NTSR1 (purple blue), and active CCK_AR (green). Side view (a) and intracellular view (b) of the overall comparison are shown. c, Structural rearrangements of key activation motifs (PIF, ERY, CWxP, and NPxxY) in CCK_AR compared to inactive ghrelin receptor and active NTSR1. NTSR1, neurotensin receptor 1.

Recognition of sulfated cholecystokinin

Sulfated CCK-8 occupies the orthosteric binding pocket composed of TM3, TM4, TM5–7 and ECL1–3 (Fig. 2 and Extended Data Figs. 2 and 3), with its C terminus inserting into the TMD bundle and the N terminus facing the extracellular vestibule (Fig. 2a). The binding pocket of CCK-8 is largely overlapped with that of other reported endogenous neuropeptides, such as neurotensin (NTS_8-13, PDB 6OS9¹⁸), angiotensin II (Ang II, PDB 6OS0³⁵), orexin B (OXB, PDB 7L1U³⁶) and arginine vasopressin (AVP, PDB 7DW9³⁷). It is noteworthy that the extracellular sides of these neuropeptides undergo remarkable conformational shifts, while their intracellular parts converge in an approximately overlapped position at the bottom of the binding pocket (Extended Data Fig. 2).

Fig. 2. Recognition of sulfated CCK-8 by CCK_AR.

a, CCK-8 sits in the orthosteric binding pocket of CCK_AR, as shown in an extracellular view (left) and side view (right). The density map of CCK-8 is shown as a magenta mesh, and CCK-8 is displayed as magenta sticks. CCK_AR is shown in green as a cutaway surface (right). ECL1 (light blue), ECL2 (lime green) and ECL3 (turquoise) are highlighted as solid surfaces. b, Detailed interactions between sulfated CCK-8 and three extracellular loops of CCK_AR. c, Effects of mutations in the receptor ligand-binding pocket on CCK-8 binding activity assessed by a radiolabeled ligand-binding assay. Data are presented as mean ± s.e.m. of three independent experiments (n = 3), except for the WT (n = 4), and conducted in triplicate. Competition curves of mutants from ECL1, TM3 and TM4 (upper), ECL2, TM5 and TM6 (middle), and ECL3 and TM7 (bottom) compared to WT CCK_AR are shown. d, Recognition of CCK-8 by the deep hydrophobic cavity beneath ECL3 of CCK_AR. e, Recognition of CCK-8 by the shallow hydrophobic cavity beneath ECL1 and ECL2 of CCK_AR. f, Recognition of CCK-8 by the bottom TMD region of CCK_AR. Key interaction residues from CCK_AR are shown as green sticks, and the receptor is shown in cartoon presentation. Polar interactions are indicated as red dashed lines.

Source data

Extended Data Fig. 2. Structure comparison of CCK-8 with other neuropeptides solved to date.

The neuropeptides are shown as a cartoon. The shift of the extracellular part of neuropeptides is highlighted as a red arrow. CCK-8 in the CCK-8–CCK_AR–G_q complex structure, magenta; Ang II, angiotensin II (PDB: 6OS0), green; OxB, orexin B (PDB: 7L1U), cyan; NTS_8-13, neurotensin 8-13 (PDB: 6OS9), purple blue; AVP, arginine vasopressin (PDB: 7DW9), salmon.

Extended Data Fig. 3. 2D interaction plot of CCK_AR recognition by sulfated CCK-8.

Residues in the ligand-binding pocket are colored in green. CCK-8 is displayed as magenta sticks. Polar interactions are indicated as red dashed lines.

It is of interest that the octapeptide CCK-8 almost completely occupies the polypeptide-binding pocket, structurally supporting the fact that it is the smallest active form of CCK isoforms. The binding modes of CCK-8 are highly conserved in all three CCK_AR–G protein complexes (all-atom r.m.s.d. of 0.71 for CCK-8 in G_q/G_s-coupled complexes, and 1.18 for CCK-8 in G_q/G_i-coupled complexes), supported by clear EM density maps (Fig. 2a and Supplementary Fig. 5). The region of ligand recognition by CCK_AR can be divided into three major parts: (1) the extracellular loops, (2) hydrophobic cavities beneath ECLs and (3) the bottom of the TMD pocket (Fig. 2a).

At the extracellular side, three ECLs are folded to embrace the N-terminal amino acids of CCK-8 (Fig. 2a). The sulfate group of ionic Y^2P interacts with the side chain of R197^ECL2. This polar interaction prompts the aromatic ring of Y^2P to form hydrophobic contacts with F185^ECL2, M195^ECL2 and the main chain of K105^ECL1, thus connecting CCK-8 to ECL1 and ECL2 (Fig. 2b and Extended Data Fig. 3). These structural observations are consistent with the previous finding that the R197^ECL2M mutation was 1,470-fold less potent than the wild-type (WT) CCK_AR¹¹. The alanine mutation of R197^ECL2 completely abolishes the binding of CCK-8, thus strongly supporting the contention that R197^ECL2 serves as a determinant to discriminate between sulfated and non-sulfated CCK (Fig. 2c and Supplementary Table 2). Likewise, poor ligand selectivity of CCK_BR may be attributed to a substitution of arginine for valine at the corresponding position (Extended Data Fig. 4). Meanwhile, M^3P, G^4P and W^5P clamp the interior surface of ECL3 (Fig. 2b).

Extended Data Fig. 4. Sequence alignment of CCK receptors.

Helical secondary structures are shown based on CCK_AR. Residues involved in ligand-binding are labeled with green triangles. Residues involved in G protein coupling are labeled with circles (orange, G_q; blue, G_s; cyan, G_i).

Two hydrophobic cavities exist below the ECLs to accommodate W^5P and M^6P (Fig. 2d,e). The side chain of W^5P is sandwiched by the side chains of I352^7.35 and R336^6.58 and buries in a deep hydrophobic pocket composed of TM6, ECL3 and TM7 (Fig. 2d). The backbone CO group of W^5P forms a hydrogen bond with R336^6.58, and its indole nitrogen atom makes another hydrogen bond with N333^6.55 (Fig. 2d and Extended Data Fig. 3), which is reported to be critical to CCK_AR activation³⁸. Alanine mutations in residues N333^6.55, R336^6.58, A343^ECL3, E344^ECL3, L347^ECL3 and S348^ECL3 completely abolish the binding of CCK-8, suggesting the key roles of these residues in CCK-8 recognition (Fig. 2c and Supplementary Table 2). In contrast to the W^5P-occupied hydrophobic pocket, M^6P sits in a relatively shallow hydrophobic cavity in the opposite direction, constituted by F107^ECL1, C196^ECL2, T118^3.29 and M121^3.32 (Fig. 2e and Extended Data Fig. 3). Mutating F107^ECL1 and residues in ECL2 and ECL3 to alanine eliminated the binding ability of CCK-8 entirely, highlighting an essential function of the three ECLs in peptide recognition (Fig. 2c and Supplementary Table 2).

At the bottom of the binding pocket, D^7P and main chain CO group of CCK-8 form a stabilizing polar interaction network with TM5 (H210^5.39), TM6 (N333^6.55 and R336^6.58) and TM7 (Y360^7.43) (Fig. 2f and Extended Data Fig. 3). The phenyl ring of F^8P makes a polar hydrogen–π interaction with Y176^4.60, and inserts into a large hydrophobic crevice composed of residues from TM3, TM4, TM5 and TM6 (Fig. 2f and Extended Data Fig. 3). Besides N333^6.55 and R336^6.58, which also interact in a polar manner with W^5P, I329^6.51 is closely related to CCK-8 binding (Fig. 2c and Supplementary Table 2).

Elucidation of the recognition mechanism of CCK-8 provides clues for therapeutic development against CCK_AR. GW-5823, CE-326597 and Glaxo-11p (Extended Data Fig. 5a) are small-molecule agonists for CCK_AR with moderate activities^10,39,40. Docking of these agonists to the CCK_AR shows that they only occupy the bottom half of the TMD binding pocket, thus lacking essential interactions with ECLs 1–3 of CCK_AR (Extended Data Fig. 5b). This structural feature may lead to a weaker activity of these small-molecule agonists relative to CCK-8. Together, our data provide a framework for understanding the mechanism of small-molecule agonist recognition and offer a template for guiding drug design targeting CCK_AR.

Extended Data Fig. 5. Molecular docking of small molecule agonists to the CCK_AR structure.

a, Chemical structures of small molecule agonists of CCK_AR. b, Comparison of the binding poses of three agonists with CCK-8. CCK-8, magenta; GW-5823, orange; CE-326597, grey; Glaxo-11p, purple blue. CCK-8 and small molecule agonists are shown as sticks. The amino acids of CCK-8 are labelled.

Overall coupling mode of CCK_AR–G protein complexes

Although all four G-protein subtypes were reported to interact with CCK_AR²⁴, only three of the CCK_AR–G protein samples (CCK_AR–G_q, CCK_AR–G_s and CCK_AR–G_i protein complexes) were obtained for high-resolution cryo-EM structure determination (Fig. 1). Structural comparison indicated that TM6 and ICL2 in CCK_AR adopt nearly identical conformations in G_q-, G_i- and G_s-coupled structures (Fig. 3a and Extended Data Fig. 6a–c). However, slightly different tilts of the Gα α5-helix were seen among the three heterotrimeric G proteins (4° for Gα_q/Gα_s and 8° for Gα_q/Gα_i; Fig. 3a). Meanwhile, the distal end of the Gα_s α5-helix moves 7 Å outward, away from the TMD core relative to the equivalent Gα_q residue (measured at the Cα atom of L^H5.25, where superscripts refer to the common Gα numbering (CGN) system⁴¹; Fig. 3a). G_q presents the largest solvent-accessible surface area (SASA, 1,492 Ų) with the receptor, compared to a G_s value of 1,293 Ų and G_i value of 1,167 Ų, consistent with a 6.6- to 20.3-fold increased potency of G_q coupling to CCK_AR in comparison to coupling with G_s and G_i (Supplementary Table 3). This finding supports the hypothesis that the size of the G-protein coupling interface may correlate with the ability of a receptor to link with different G proteins^21,22. In addition, coupling of different G-protein subtypes exhibits distinct effects on CCK-8 binding. Compared to G_s or G_i proteins, G_q coupling increases the binding affinity of CCK-8 (Supplementary Table 3), consistent with the increased binding activity of isoproterenol against β_2AR in the presence of G_s protein¹⁶. This finding indicates an allosteric modulation effect of G_q protein on CCK-8 binding, supporting the positive cooperativity between agonists and G proteins⁴².

Fig. 3. Structural comparison of TM6 and the α5-helix between CCK_AR–G protein complexes and representative G_s-, G_q- and G_i-coupled GPCR structures, in two different views.

a, Structural comparison of CCK_AR–G_q, CCK_AR–G_s and CCK_AR–G_i complexes. A 7-Å movement of the distal end of the Gα_s α5-helix (α5H) relative to that of Gα_q and a swing of the Gα α5-helix are highlighted as red arrows. b, Structural comparison of CCK_AR–G_s with β_2AR–G_s and A_2AR–G_s complexes. Red arrows indicate an 11–12-Å displacement of TM6 and a 9–11° swing of the Gα α5-helix of G_s-coupled CCK_AR relative to G_s-coupled β_2AR and A_2AR. c, Structural comparison of CCK_AR–G_i with the NTSR1–G_i and M₂R–G_o complexes. A 4-Å inward displacement of TM6 and a 3-Å Gα_i α5-helix shift of G_q-coupled CCK_AR in comparison to G_o-coupled M₂R are indicated as red arrows. d, Structural comparison of CCK_AR–G_q with 5-HT_2AR–G_q and M₁R–G₁₁ complexes. A 2-Å upward movement of Gα_q of G_q-coupled CCK_AR compared to G_q-coupled 5-HT_2AR and a 28° rotation relative to G₁₁-coupled M₁R are highlighted as red arrows. The complex structures are aligned based on TM2–TM4 of the receptors. β_2AR–G_s, A_2AR–G_s, NTSR1–G_i, M₂R–G_o, 5-HT_2AR–G_q and M₁R–G₁₁ structures (PDB 3SN6, 5G53, 6OS9, 6OIK, 6WHA and 6OIJ) are colored in gray, marine, purple blue, dark brown, magenta and yellow, respectively.

Extended Data Fig. 6. The interface between CCK_AR ICL2 and different G proteins.

Detailed interaction between the receptor and Gα_q (a), Gα_s (b), and Gα_i (c) are shown. Side chains of related residues are shown as sticks.

In addition, comparisons of these three complex structures to previously reported G-protein-coupled class A GPCRs reveal the different extent of TM6 displacement and the concomitant shift of the Gα α5-helix (Fig. 3b–d). TM6 of CCK_AR in all three G-protein complexes displays an 11–12-Å (measured at the Cα atom of the residue at position 6.27) smaller outward displacement compared to G_s-coupled GPCRs, which translates into a notable swing of the Gα α5-helix in the same direction (9–11° relative to G_s-coupled β_2AR and A_2AR as measured at the Cα atom of Y^H5.23). This smaller displacement of TM6 is contrary to the previous assumption that TM6 of G_s-coupled GPCRs undergoes a notable outward movement, thus opening a larger cytoplasmic pocket to accommodate bulkier residues at the distal end of the Gα_s α5-helix relative to G_i/o-coupled receptors^21,43. To avoid a potential clash with TM6, the distal end of the Gα_s α5-helix in the CCK_AR–G_s complex stretches away from the TMD core and inserts into the crevice between the TM6 and TM7–helix 8 joint. This featured conformation of the Gα_s α5-helix in the CCK_AR–G_s complex is unique compared to that in structures of the G_s-coupled β_2AR and A_2AR, supporting the complexity of the GPCR–G protein coupling mechanism (Fig. 3b).

TM6 and the Gα α5-helix of CCK_AR–G protein complexes display similar conformational changes to other G_i- and G_q-coupled GPCRs, such as the G_i-coupled NTSR1 and the G_q-coupled 5-HT_2AR (Fig. 3c,d). TM6 of the CCKA_R–G_i protein complex is highly overlaid with that of G_i-coupled NTSR1, while the cytoplasmic end of TM6 shows a 4-Å smaller outward displacement compared to that of G_o-coupled M₂R (Fig. 3c). On the G-protein side, the α5-helix of Gα_i in the CCK_AR–G_i complex shows a nearly overlapped conformation with that of the NTSR1–G_i complex. In contrast, it exhibits a 3-Å (measured at the Cα atom of Y^H5.23) shift away from TM6 relative to that of G_o-coupled M₂R (Fig. 3c). Structural comparison of G_q-coupled CCK_AR with G_q/G₁₁-coupled GPCRs demonstrates a 2-Å (measured at the Cα atom of Y^H5.23) upward shift toward the cytoplasmic cavity in comparison to the G_q-coupled 5-HT_2AR and a 28° rotation away from TM6 relative to G₁₁-coupled M₁R (Fig. 3d).

Interactions of the ‘wavy hook’ of CCK_AR–G protein complexes

The ‘wavy hook’ at the extreme C terminus of the Gα α5-helix is thought to be one of the coupling specificity determinants for G proteins^44,45, and undergoes distinct conformational rearrangements among the three CCK_AR–G protein complexes (Fig. 3a).

A structural comparison of the interaction interface between the receptor cytoplasmic cavity and the Gα ‘wavy hook’ reveals distinct features of CCK_AR–G protein coupling. Well-defined densities of Gα-protein ‘wavy hook’ residues allow for detailed structural analyses except for residues at the –1 position. The L(–2)^H5.25 in the α5-helix is highly conserved across the G-protein families and plays a pivotal role in G-protein coupling. Both L358^H5.25 in Gα_q and L353^H5.25 in Gα_i hydrophobically interact with residues in TM3 and TM6 (R139^3.50, I143^3.54, V311^6.33 and L315^6.37; Fig. 4a,b). Owing to the notable displacement of the Gα_s C terminus, L393^H5.25 in Gα_s moves 7 Å outward away from the TMD core relative to the equivalent Gα_q residue (Fig. 3a), repositioning it in a hydrophobic subpocket formed by M314^6.36 and M373^7.56 (Fig. 4c). In contrast to L(–2)^H5.25, residues at positions H(–3)^5.24, H(–4)^5.23 and H(–5) ^5.22 are less conserved. N357(–3)^H5.24 in Gα_q makes a hydrogen bond with the backbone CO group of Y370^7.53 (Fig. 4a). As a result of the replacement of Gα_i G352(–3)^H5.24 and the repositioning of Gα_s E392(–3)^H5.24, the corresponding hydrogen bond is absent in CCK_AR–G_i and CCK_AR–G_s complex structures. Additionally, Y356(–4)^H5.23 in Gα_q forms extensive interactions with the receptor cytoplasmic cavity by making hydrogen bonds with R139^3.50 and Q153^ICL2 (Fig. 4d). By contrast, C351(–4)^H5.23 in Gα_i only forms a weak hydrogen bond with R139^3.50 via its backbone CO group (Fig. 4e). Y391(–4)^H5.23 in Gα_s exhibits limited hydrophobic and van der Waals interactions with residues in TM2 and TM3 (T76^2.39, R139^3.50 and A142^3.53; Fig. 4f). Furthermore, both E355(–5)^H5.22 in Gα_q and D350(–5)^H5.22 in Gα_i form salt bridges with R376^8.49 in CCK_AR, while Q390(–5)^H5.22 in Gα_s disfavors the formation of the corresponding electrostatic interaction (Fig. 4d–f). To understand the ‘wavy hook’-mediated G-protein selectivity, we displaced the amino acids (H5.22–H5.25) in the Gα_q subunit with the corresponding ones in the Gα_s and Gα_i subunits. Bioluminescence resonance energy transfer (BRET) assay results show that the Gα_i displacement has no impact on CCK_AR–G protein coupling compared to the WT Gα_q subunit. However, partial (E355Q or N357E) or complete Gα_s substitution remarkably decreased the G-protein coupling activity of CCK_AR (Fig. 4g). These results indicate that the ‘wavy hook’ may play a crucial role in the coupling selectivity of CCK_AR with G_q over the G_s protein.

Fig. 4. Distinct interaction patterns of residues from the ‘wavy hook’ motif.

a–c, Details of the interaction between CCK_AR and L358^H5.25 and N357^H5.24 of the Gα_q subunit (a), L353^H5.25 of the Gα_i subunit (b) and L393^H5.25 of the Gα_s subunit (c). d–f, Details of the interaction between CCK_AR and Y356^H5.23 and E355^H5.22 of the Gα_q subunit (d), C351^H5.23 and D350^H5.22 of the Gα_i subunit (e) and Y391^H5.23 and Q390^H5.22 of the Gα_s subunit (f). Hydrogen bonds and salt bridges are indicated as red dashed lines. g, BRET assay evaluating the effects of ‘wavy hook’ substitutions on CCK_AR–G protein coupling. The ‘wavy hook’ residues of the Gα_q subunit were displaced by the corresponding residues in the Gα_s and Gα_i subunits. Data are shown as mean ± s.e.m. of three independent experiments (n = 3), conducted in triplicate. All data were analyzed by one-way analysis of variance Dunnett multiple comparisons test (P = 1, P = 0.9827, P = 0.7521, P = 0.9994, P = 0.7421, P < 0.0001, P < 0.0001 and P = 0.2078 from top to bottom, ***P < 0.001, ****P < 0.0001 versus WT).

Source data

Contribution of CCK_AR ICL3 to G_q coupling selectivity

In the CCK_AR–G_q protein complex structure, CCK_AR displays a comparable length of TM5 relative to the M₁R–G₁₁ complex¹⁹. However, the cytoplasmic end of the CCK_AR TM5 exhibits an 8-Å outward bend (measured at the Cα atoms of A^5.73), which prevents it from interacting with the Gα_q subunit (Fig. 5a). Instead, the ICL3 inserts into the cleft between TM5 of CCK_AR and the α5-helix of the Gα_q subunit (Fig. 5a). Compared to L225^5.75 in M₁R, I296^ICL3 in CCK_AR interacts with the same hydrophobic patch formed by the side chains of Y325^S6.02, F339^H5.06 and A342^H5.09 in the Gα_q subunit, but is buried deeper to create more closely packed hydrophobic contacts (Fig. 5a,b). These hydrophobic interactions are critical to CCK_AR–G_q coupling, as evidenced by our BRET analysis showing that the I296^ICL3G mutation significantly weakens G_q coupling to CCK_AR but has no impact on G_s and G_i coupling (Fig. 5c and Supplementary Table 4). This hydrophobic patch, which lies on the outer surface, may be unique for the G_q/11 subunit. The equivalent residues in the Gα_s and Gα_i subunits are polar or charged residues, for which it would be energetically unfavorable to form hydrophobic interactions (Extended Data Fig. 7a–d). Indeed, this unconventional ICL3–G_q interaction is not seen in the structures of G_s- and G_i/o-coupled CCK_ARs (Fig. 3b,c). Together, our findings offer structural evidence on the possible role of ICL3 in CCK_AR–G_q coupling preference. Hydrophobic residues on the inner surface of the ICL3 loop of CCK_AR or the extended TM5 of M₁R may represent a common feature of G_q/11-coupled GPCRs.

Fig. 5. Interaction between the ICL3 loop of CCK_AR and the Gα_q subunit.

a, I296^ICL3 of CCK_AR and L225^5.75 of M₁R occupy the same hydrophobic subpocket of the Gα subunit. The Gα_q subunit is shown as a surface presentation by hydrophobicity (hydrophobic surface in red). An 8-Å outward bend of TM5 of CCK_AR relative to that of M₁R is highlighted by a black arrow. b, Detail of the interactions between I296^ICL3(CCK_AR)/L225^5.75(M₁R) and the hydrophobic patch comprising Y325^S6.02, F339^H5.06 and A342^H5.09 of the Gα_q and Gα₁₁ subunits. c, BRET assay showing that the I296G mutation decreases the association rate of CCK_AR with the G_q heterotrimer. Data are shown as mean ± s.e.m. of three independent experiments (n = 3), conducted in triplicate.

Source data

Extended Data Fig. 7. Comparison of the hydrophobic patch in Gα_q subunit to the corresponding sites in other G proteins.

a, Sequence alignment of S6.02, H5.06, and H5.09 from Gα_q, Gα_s, and Gα_i subunits. Residues at positions S6.02, H5.06, and H5.09 comprise the hydrophobic patch to interact with CCK_AR ICL3. b-d, Surface presentation of the patch by hydrophobicity. Side chains of residues at positions S6.02, H5.06, and H5.09 in Gα_q (b), Gα_s (c), and Gα_i (d) subunits are shown.

Discussion

As the largest family of cell surface receptors, GPCRs have more than 800 members but only couple to four G-protein subtypes. Specific GPCR signaling requires the receptor to couple with either a single or multiple G-protein subtypes^45–47. Thus, one of the main questions is how does a given GPCR select a G-protein subtype for downstream signal transduction. The critical G-protein determinants of selectivity vary widely for different receptors that couple to specific G proteins. It is thought that G_s- or G_q-coupled receptors are relatively promiscuous and to some extent couple to G_i1 (ref. ²²). However, G_i-coupled receptors are more selective²². The minor outward movement of TM6 contributes to such a superior G_i coupling selection in comparison to that of G_s (refs. ^{17,23,44,48,49}). Although proven to be promiscuous, G_q-coupled receptors tend to adopt an active conformation similar to that of G_i-coupled GPCRs, reflecting the complexity of the GPCR–G protein coupling mechanism^19,20. Because CCK_AR has the ability to couple with different G-protein subtypes, it stands out as a suitable model for studying the promiscuity of G-protein coupling. In this article, we show that TM6 of CCK_AR undergoes a similar outward displacement relative to G_i/o-coupled (NTSR1 and M₂R) and G_q/11-coupled GPCRs (5-HT_2AR and M₁R), but has a smaller shift relative to G_s-coupled GPCRs (β_2AR and A_2AR). CCK_ARs share almost identical conformations, whereas G_q, G_s and G_i proteins vary in distinct orientations, producing different sizes of receptor–G protein interface. The predominant coupling to G_q by CCK_AR can be explained by its largest interface of the three CCK_AR–G protein complexes. Structural comparison of the three CCK_AR–G protein complexes reveals that ‘wavy hook’ residues of the Gα α5-helix and ICL3 of the receptor are important for the coupling promiscuity. In addition, detailed inspections disclose structural clues relative to the recognition mechanism of sulfated CCK-8 by CCK_AR, in which R197^ECL2 is a major determinant. Together, our structures provide a framework for better understanding ligand recognition as well as G-protein coupling selectivity and promiscuity by CCK_AR.

Methods

Expression and purification of CCK_AR–G protein complexes

The WT CCK_AR (residues 1–428) was used for cryo-EM studies. Full-length CCK_AR complementary DNA was cloned into a modified pFastBac vector (Invitrogen) containing a hemagglutinin (HA) signal sequence followed by an 8× histidine tag, a double-maltose binding protein tag and a tobacco etch virus (TEV) protease site before the receptor sequence using homologous recombination (using a CloneExpress One Step Cloning Kit, Vazyme; Supplementary Fig. 4a). The N-terminal 1–29 amino acids of Gα_q were replaced by the equivalent residues of Gα_i1 to facilitate scFv16 binding¹⁹. An engineered Gα_s construct was generated based on mini-Gα_s³¹. The N-terminal 1–18 amino acids and the α-helical domain of Gα_s were replaced by human Gα_i1, thus providing binding sites for scFv16 and Fab-G50, respectively^17,19. Additionally, human Gα_i1 with two dominant-negative mutations (G203A and A326S³²) was used to assemble a stable GPCR–G_i protein complex. These two cognate mutations also exist in engineered Gα_q and Gα_s (Supplementary Fig. 4b). Receptor, rat H6-Gβ, bovine Gγ and the specific Gα subunit were co-expressed in Spodoptera frugiperda (sf9) insect cells (Invitrogen) as previously described⁵⁰. In addition, GST-Ric-8A (a gift from B. Kobilka) was applied to improve the expression of Gα_q.

ScFv16 was applied to improve the protein stability of CCK_AR–G_q and CCK_AR–G_i complex samples. The monomeric scFv16 was prepared as previously reported⁵¹. Cell pellets of the co-expression culture were thawed and lysed in 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 5 mM MgCl₂ and 10 mM CaCl₂ supplemented with EDTA-free protease inhibitor cocktail (TargetMol). CCK_AR–G protein complexes were assembled at room temperature (r.t.) for 1 h by the addition of 10 μM CCK-8 (GenScript) and 25 mU ml⁻¹ apyrase. The lysate was then solubilized in 0.5% lauryl maltose neopentyl glycol (LMNG), 0.1% cholesteryl hemisuccinate TRIS salt (CHS), and the soluble fraction was purified by nickel affinity chromatography (Ni Smart Beads 6FF, SMART Lifesciences). In the case of CCK_AR–G_i and CCK_AR–G_q complexes, a three-molar excess of scFv16 was added to the protein elute. The mixture was incubated with amylose resin for 2 h at 4 °C. The excess G protein and scFv16 were washed with 20 column volumes of 20 mM HEPES, pH 7.4, 100 mM NaCl, 10% glycerol, 0.01% LMNG, 0.002% CHS and 2 μM CCK-8. TEV protease was then included to remove the N-terminal fusion tags of CCK_AR. After 1 h of incubation at r.t., the flow-through was collected, concentrated and injected onto a Superdex 200 10/300 column equilibrated in buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 0.00075% LMNG, 0.00025% glycol-diosgenin (GDN), 0.0002% CHS and 10 μM CCK-8. The monomeric complex peak was collected and concentrated to ~5 mg ml⁻¹ for cryo-EM studies.

Cryo-electron microscopy grid preparation and image collection

For preparation of cryo-EM grids, 2.5 µl of each purified CCK_AR–G protein complex was applied individually onto glow-discharged holey carbon grids (Quantifoil, Au300 R1.2/1.3) in a Vitrobot chamber (FEI Vitrobot Mark IV). The chamber was set to 100% humidity at 4 °C. Extra samples were blotted for 2 s and vitrified by plunging into liquid ethane. Grids were stored in liquid nitrogen for condition screening and data collection usage.

Automatic data collection of CCK-8–CCK_AR–G protein complexes was performed on an FEI Titan Krios system at 300 kV. The microscope was operated with a nominal magnification of ×81,000 in counting mode, corresponding to a pixel size of 1.045 Å for the micrographs. A total of 5,415 videos for the dataset of the CCK-8–CCK_AR–G_q–scFv16 complex, 5,008 for the dataset of the CCK-8–CCK_AR–G_s complex and 4,811 for the dataset of the CCK-8–CCK_AR–G_i–scFv16 complex were collected, respectively, by a Gatan K3 Summit direct electron detector with a Gatan energy filter (operated with a slit width of 20 eV; GIF) using SerialEM software. The images were recorded at a dose rate of ~26.7 e Å⁻² s⁻¹ with a defocus ranging from −0.5 to −3.0 μm. The total exposure time was 3 s and intermediate frames were recorded in intervals of 0.083 s, resulting in a total of 36 frames per micrograph.

Image processing and map reconstruction

Image stacks were subjected to beam-induced motion correction and aligned using MotionCor 2.1. Contrast transfer function (CTF) parameters were estimated by Ctffind4. Data processing was performed using RELION-3.0⁵². Micrographs with measured resolution worse than 4.0 Å and micrographs imaged within the carbon area were discarded, generating 3,806 micrographs for the CCK-8–CCK_AR–G_q–scFv16 dataset, 4,963 for the CCK-8–CCK_AR–G_s dataset and 4,543 for the CCK-8–CCK_AR–G_i–scFv16 dataset for further data processing. For particle selection, two-dimensional (2D) and 3D classifications were performed on a binned dataset with a pixel size of 2.09 Å. About 2,000 particles were manually selected and subjected to 2D classification. Representative averages were chosen as a template for particle autopicking. The autopicking process produced 3,405,355 particles for the CCK-8–CCK_AR–G_q–scFv16 complex, 4,680,972 for the CCK-8–CCK_AR–G_s complex and 4,270,010 for the CCK-8–CCK_AR–G_i–scFv16 complex, which were subjected to reference-free 2D classifications to discard bad particles. Initial reference map models for 3D classification were generated by Relion using representative 2D averages. For the CCK-8–CCK_AR–G_q–scFv16 complex, the particles selected from 2D classification were subjected to six rounds of 3D classification, resulting in a single well-defined subset with 555,628 particles. For the CCK-8–CCK_AR–G_s complex, particles resulting from 2D classification were subjected to five rounds of 3D classification, resulting in two well-defined subsets with 499,924 particles. For the CCK-8–CCK_AR–G_i–scFv16 complex, particles selected from 2D classification were subjected to seven rounds of 3D classifications, resulting in two well-defined subsets with 140,602 particles. Further 3D refinement, CTF refinement, Bayesian polishing and DeepEnhancer processing generated density maps with an indicated global resolution of 2.9 Å for the CCK-8–CCK_AR–G_q–scFv16 complex, 3.1 Å for the CCK-8–CCK_AR–G_s complex and 3.2 Å for the CCK-8–CCK_AR–G_i–scFv16 complex, respectively, at a Fourier shell correlation of 0.143.

Model building and refinement

For the CCK_AR–G_q complex, the initial G_q protein and scFv16 model were adopted from the cryo-EM structure of the M₁R–G₁₁ protein complex (PDB 6OIJ)¹⁹. The initial CCK_AR model was generated by an online homology model building tool⁵³. All models were docked into the EM density map using Chimera⁵⁴, followed by iterative manual adjustment and rebuilding in COOT⁵⁵ and ISOLDE⁵⁶, and real-space refinement using Phenix programs⁵⁷. The model statistics were validated using Phenix comprehensive validation. A model of the refined CCK_AR from the CCK_AR–G_q complex was used for the other two complexes. Models from PTH1R–G_s (PDB 6NBF) and FPR2–G_i (PDB 6OMM) were used as templates for the model building of G_s in the CCK_AR–G_s complex and G_i1–scFv16 in the CCK_AR–G_i complex, respectively. The fitted models were then built in the same way as the CCK_AR–G_q complex. The final refinement statistics are provided in Supplementary Table 1. All figures were prepared using PyMol and Chimera software.

Radiolabeled ligand-binding assay

The WT or mutant CCK_ARs were transiently transfected into HEK 293T/17 cells (purchased from the Cell Bank at the Chinese Academy of Sciences), which were cultured in a poly-d-lysine-coated 96-well plate. After 24 h, the cells were washed twice and incubated with blocking buffer (Dulbecco’s modified Eagle medium (DMEM) supplemented with 33 mM HEPES, and 0.1% (wt/vol) bovine serum albumin (BSA), pH 7.4) for 2 h at 37 °C. After three washes with ice-cold phosphate-buffered saline (PBS), the cells were treated by a constant concentration of ¹²⁵I-CCK-8 (40 pM, PerkinElmer) plus eight different doses of CCK-8 (1 pM to 10 μM) for 3 h at r.t. Cells were washed three times with ice-cold PBS and lysed by 50 μl of lysis buffer (PBS supplemented with 20 mM Tris-HCl and 1% (vol/vol) Triton X-100, pH 7.4). Subsequently, the plates were counted for radioactivity (counts per minute) in a scintillation counter (MicroBeta² plate counter, PerkinElmer) using 150 μl of scintillation cocktail (OptiPhase SuperMix, PerkinElmer).

G-protein dissociation assay

G-protein dissociation was monitored by BRET experiments performed as previously reported⁵⁸. Briefly, a C-terminal fragment of the G-protein-coupled receptor kinase 3 (GRK3ct) fused to a luciferase serves as a BRET donor. Gβγ dimer is labeled with the fluorescent protein Venus, a BRET acceptor. Upon G-protein heterotrimer activation, free Gβγ-Venus is released and binds to membrane-associated GRK3ct-luciferase, leading to an increased signal detectable by BRET.

HEK 293T/17 cells were seeded onto a 10 μg ml⁻¹ Matrigel-coated six-well plate (1 × 10⁶ cells per well). After 4 h of culture, WT or mutant CCK_AR (0.84 μg), Gα (Gα_q, Gα_s and Gα_i, 2.1 μg each), Gβ (0.42 μg), Gγ (0.42 μg) and GRK (0.42 μg) were transiently transfected with Lipofectamine LTX reagent (Invitrogen). At 24 h post-transfection, cells were washed once with DMEM medium (no phenol red) and detached by EDTA. Cells were then collected by centrifugation at 1,000 r.p.m. for 5 min and resuspended in DMEM medium. Approximately 75,000 cells per well were distributed in 96-well flat-bottomed white microplates (PerkinElmer). The NanoBRET substrate (furimazine, 25 μl per well, Promega) was added, and the BRET signal (535 nm/475 nm ratio) was determined using an EnVision multilabel plate reader (PerkinElmer). The average baseline value recorded before CCK-8 stimulation was subtracted from BRET signal values.

NanoBiT G-protein recruitment assay

The recruitment of CCK_AR to G_i protein was detected in sf9 cells using the NanoBiT method as previously reported⁵⁹. Briefly, the LgBiT fragment of NanoBiT luciferase was fused to the C terminus of CCK_AR. SmBiT was fused to the C terminus of the Gβ subunit with a 15-amino-acid flexible linker. CCK_AR-LgBiT, Gα_i1, SmBiT-fused human Gβ1 and human Gγ2 were co-expressed in sf9 insect cells. Cell pellets were collected by centrifugation after infection for 48 h. The cell suspension was dispensed in a 96-well plate (64,000 cells per well) at a volume of 80 μl diluted in the assay buffer (Hanks’ balanced salt solution buffer supplemented with 10 mM HEPES, pH 7.4) and incubated for 30 min at 37 °C. The cells were then reacted with 10 μl of 50 mM coelenterazine H (Yeasen) for 2 h at r.t. The luminescence signal was measured using an EnVision plate reader (PerkinElmer) at 30-s intervals (25 °C). The baseline was measured before CCK-8 addition for eight intervals, and the measurements continued for 20 intervals following ligand addition. Data were corrected to baseline measurements and the results were analyzed using GraphPad Prism 8.0 (Graphpad Software).

NanoBiT G-protein dissociation assay

G_s activation was measured by a NanoBiT dissociation assay. G-protein NanoBiT split luciferase constructs were generated by fusing the LgBiT in Gα_s and the SmBiT to Gγ (a gift from A. Inoue, Tohoku University) as previously reported⁶⁰. In brief, HEK 293T/17 cells were plated in 10-cm plates at a density of 3 × 10⁶ cells per plate. After 24 h, cells were transfected with 1.62 μg receptor plasmids, 0.81 μg Gα_s-LgBiT, 4.1 μg Gβ and 4.1 μg SmBiT-Gγ using Lipofectamine LTX reagent (Invitrogen). The transiently transfected cells were then seeded into poly-d-lysine-coated 96-well plates (50,000 cells per well) and grown overnight before incubation in assay buffer. Measurement of the luminescence signal was identical to the steps described above.

Surface expression assay

HEK 293T/17 cells were seeded into a six-well plate and incubated overnight. After transient transfection with WT or mutant plasmids for 24 h, the cells were collected and blocked with 5% BSA in PBS at r.t. for 15 min and incubated with primary anti-Flag antibody (1:300, Sigma-Aldrich) at r.t. for 1 h. The cells were then washed three times with PBS containing 1% BSA followed by 1 h of incubation with donkey anti-mouse Alexa Fluor 488-conjugated secondary antibody (1:1,000, Thermo Fisher) at 4 °C in the dark. After three washes, the cells were resuspended in 200 µl of PBS containing 1% BSA for detection in a NovoCyte flow cytometer (ACEA Biosciences) utilizing laser excitation and emission wavelengths of 488 nm and 519 nm, respectively. For each assay point, ~15,000 cellular events were collected, and the total fluorescence intensity of the positive expression cell population was calculated. The gating strategy and the method for the calculation of expression are shown in Supplementary Fig. 6.

Molecular docking

Before docking, hydrogens were added to CCK_AR and the whole system coordinates were optimized with a pH of 7.0. A grid file was then generated on the peptide pocket in our G_q-coupled CCK_AR structure. Small-molecule ligands Glaxo-11p, GW-5823 and CE-326597 were prepared in the OPLS3 force field with a pH of 7.0 to generate 3D structures. Finally, glide docking with standard precision was applied to all ligands and the structures with the best docking score were picked as outputs.

Statistics

All functional study data were analyzed using Prism 8 (GraphPad) and are shown as the mean ± s.e.m. from at least three independent experiments. Concentration–response curves were evaluated with a three-parameter logistic equation. Significance was determined with either a two-tailed Student’s t-test or one-way analysis of variance Dunnett multiple comparisons test, and P < 0.05 was considered statistically significant.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this Article.

Online content

Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41589-021-00841-3.

Source data

Source Data Fig. 2 ^{(16.2KB, xlsx)}

Statistical source data.

Source Data Fig. 4 ^{(11.7KB, xlsx)}

Statistical source data.

Source Data Fig. 5 ^{(10.1KB, xlsx)}

Statistical source data.

Author contributions

Q.L. screened the expression constructs, optimized the CCK_AR–G protein complexes, prepared the protein samples for final structure determination, participated in cryo-EM grid inspection and data collection, built and refined the structure models, prepared the constructs for functional assays, analyzed the structures and prepared the figures and wrote the initial manuscript. Y.Z. performed cryo-EM grid preparation, data collection and structure determination and participated in protein sample optimization and figure and manuscript preparation. T.I.C. helped build and refine the structure model. X.H. performed the molecular docking. J.D. and W.Y. designed G-protein constructs. F.Z. participated in data analysis. B.W. and Q.Z. supervised research. H.E.X. conceived and supervised the project, analyzed the structures and initiated collaborations with M.-W.W., and supervised Q.L., Y.Z., F.Z., J.D. and W.Y. M.-W.W. and D.Y. supervised X.C., A.D. and C.Y. in G-protein assay development and data analysis. M.-W.W. participated in manuscript writing. Y.J. supervised the studies, performed the structural analysis, prepared the figures and wrote the manuscript with input from all co-authors.

Data availability

Density maps and structure coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) with accession codes EMD-31387 and 7EZH for the CCK-8–CCK_AR–G_i–scFv16 complex, EMD-31388 and 7EZK for the CCK-8–CCK_AR–G_s complex, and EMD-31389 and 7EZM for the CCK-8–CCK_AR–G_q–scFv16 complex. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Extended data

is available for this paper at 10.1038/s41589-021-00841-3.

Supplementary information

The online version contains supplementary material available at 10.1038/s41589-021-00841-3.