Molecular basis of ligand recognition and activation of the human succinate receptor SUCR1

Dear Editor,

Succinic acid, once considered merely a tricarboxylic acid cycle intermediate,¹ has been recognized for its significant role in influencing mitochondrial reactive oxygen species homeostasis.² This is largely mediated through the G protein-coupled succinate receptor (SUCR1, also known as GPR91), which has emerged as a vital link connecting metabolic status to a myriad of physiological and pathological processes. SUCR1 is intricately involved in the regulation of blood pressure,³ angiogenesis,⁴ inflammation,⁵ and has been implicated in the pathogenesis of liver fibrosis,⁶ hypertension, and rheumatic arthritis.⁶ These multifaceted roles highlight the receptor’s potential as a promising therapeutic target for a wide spectrum of diseases. However, the molecular mechanisms underlying SUCR1’s activation by various ligands and its subsequent interaction with the inhibitory G protein (G_i) have remained elusive, hindering our comprehensive understanding of its broad physiological significance and therapeutic potential.

To address this knowledge gap, we determined three cryo-electron microscopy (cryo-EM) structures of the human SUCR1–G_i complexes with the endogenous succinic acid, maleic acid, and the structurally related compound 31 which is a SUCR1 agonist with higher affinity,⁷ using combinatorial approaches of BRIL fusion, G_i engineering, and stabilizing antibody scFv16, which had previously been used successfully in solving several GPCR–G protein complexes (Supplementary information, Fig. S1).^8,9 Succinic acid-, maleic acid-, and compound 31-bound SUCR1–G_i complexes were solved at global resolutions of 2.75 Å, 2.69 Å, and 2.48 Å, respectively (Fig. 1a; Supplementary information, Figs. S2–S4 and Table S1).

Fig. 1. Molecular mechanisms underlying ligand recognition and activation of SUCR1 receptor.

a Cryo-EM density map and cartoon presentation of the SUCR1–G_i complex. b Structural alignment of SUCR1 with GPR84, highlighting the overall structural features of SUCR1. c, d Detailed interactions between SUCR1 and succinic acid (c) and maleic acid (d), showing key binding residues. e, f Dose-response curves of succinic acid (e) and maleic acid (f) in activating the mutated SUCR1. Data are means ± SEM from 3 independent experiments. g, h Interactions between SUCR1 and the “succinic acid” motif (g) and the “two-ring” motif (h) of compound 31, illustrating critical binding residues. i, j Dose-response curves of compound 31 in activating SUCR1 variants which harbor mutations in the binding regions of the “succinic acid” motif (i) and the “two-ring” motif (j) of compound 31. Data are means ± SEM from 3 independent experiments. k, l Structural alignments of SUCR1 complexes in side view (k) and extracellular view (l). m Superposition of the active SUCR1 structure (green) with the inactive SUCR1 structure (gray, PDB: 6RNK), highlighting conformational changes in the intracellular ends of TM6 and TM7 upon receptor activation. n A hydrophobic packing involving L102^3.32, L106^3.36, F241^6.44, F245^6.48, and F285^7.43 contributes to SUCR1 activation.

In all structures, SUCR1 adopts an active-like state typical of GPCR–G protein coupling. The overall structure of SUCR1 is similar to that of the medium-chain fatty acid receptor, GPR84 (PDB: 8J18)¹⁰ with root mean square deviation (RMSD) of 0.36 Å for the Cα atoms of SUCR1 (residues 11–309) and GPR84 (residues 8–395). In both receptors, the extracellular loop 2 (ECL2) forms a lid over the ligand-binding pocket (Fig. 1b). Due to the smaller ligands and more compact ligand-binding pockets, the SUCR1 structure appears leaner compared to other class A GPCRs, including GPR84, with the extracellular domains of transmembrane helices 1, 5, and 6 (TM1, TM5, and TM6) shifted inward to the ligand-binding pocket (Fig. 1b). In addition, structural comparisons indicate that the TM1 of SUCR1 is significantly longer than that of GPR84, which may contribute to the enhanced stability of the SUCR1 receptor (Fig. 1b).

In the succinic acid-bound SUCR1 structure, succinic acid is securely held within a binding pocket formed by TM1, TM2, TM3, TM7, and ECL2, exhibiting a shape resembling the letter “C”. The pocket is highly electrostatically positive, accommodating the negatively charged succinic acid. The two carboxylic acid groups of succinic acid are oriented towards the extracellular side of the ligand-binding pocket, forming hydrogen bonds with Y30^1.39 and Y83^2.64, and ionic bonds with R99^3.29 and R281^7.39, respectively (Fig. 1c). Additionally, Y30^1.39 forms a hydrogen bond with R281^7.39, stabilizing the hydrophilic environment of the binding pocket (Fig. 1c). As mentioned above, ECL2 is located on the top of the succinic acid and covers the entry of the ligand-binding pocket. Simultaneously, the alkyl portion in the middle of the ligand predominantly orients itself towards the bottom of the ligand-binding pocket, establishing hydrophobic interactions with residues L79^2.60, L102^3.32, and F285^7.43 (Fig. 1c).

Maleic acid is a structural analogue of succinic acid, bearing an alkene moiety in the alkyl portion. In the maleic acid-bound SUCR1 structure, maleic acid adopts a near-identical binding pose to succinic acid within the ligand-binding pocket. Specifically, the carboxylic acid groups on the same side form polar interactions with residues from SUCR1, including Y30^1.39, Y83^2.64, R99^3.29, H103^3.33, and R281^7.39 (Fig. 1d). The alkene of maleic acid also forms hydrophobic interactions with L79^2.60, L102^3.32, and F285^7.43 similar to succinic acid (Fig. 1d). Alanine mutations of binding pocket residues, including Y30^1.39, L79^2.60, Y83^2.64, R99^3.29, and R281^7.39, significantly impaired the activation potential of both succinic acid and maleic acid, underscoring their essential roles in SUCR1 function (Fig. 1e, f; Supplementary information, Fig. S5 and Tables S2, S3). These findings highlight the intricate molecular interactions that govern SUCR1 regulation and activation by succinic acid and maleic acid.

Compound 31 is a synthetic small molecule that activates SUCR1 with high potency.⁷ In this study, we found that compound 31 activates SUCR1 by G_i signaling pathway with ~100-fold higher potency than succinic acid and maleic acid (Supplementary information, Fig. S5). The structure of compound 31 is composed of part 1 “succinic acid” motif and part 2 “two-ring” motif (Supplementary information, Fig. S6a). Comparison of the compound 31-bound SUCR1 structure with the succinic acid-bound structure reveals a deflection of the R99^3.29 side chain, resulting in the formation of an extended binding pocket (Supplementary information, Fig. S6b). In the ligand-binding pocket of the compound 31-bound SUCR1 structure, the part 1 “succinic acid” motif of compound 31 partially aligns with the position of succinic acid and maleic acid (Supplementary information, Fig. S6b), forming polar interactions with surrounding amino acids such as Y30^1.39, Y83^2.64, H103^3.33, and R281^7.39, and nonpolar interactions with some amino acids such as L79^2.60, L102^3.32, F285^7.43(Fig. 1g). The binding regions of succinic acid, maleic acid, and the part 1 “succinic acid” motif of compound 31 in SUCR1 partially overlap with those identified in the recent molecular dynamics simulation studies,¹¹ including interactions with R^3.29 and R^7.39. However, the part 2 “two-ring” motif of compound 31 binds to a newly discovered region that extends outward from the binding pocket, with the pyridine ring constrained by L79^2.60, W88^ECL1, N98^3.28, and R99^3.29 (Fig. 1h). In addition, the benzene ring in compound 31 is clamped by Y83^2.64 and W88^ECL1 (Fig. 1h). The formation of interactions with surrounding SUCR1 residues by the part 2 “two-ring” motif may account for the significantly stronger potency of compound 31 in activating SUCR1 than succinic acid and maleic acid. Mutation of Y30^1.39, L79^2.60, N87^ECL1, N98^3.28, R99^3.29, and R281^7.39 in SUCR1 reduced compound 31-induced signaling responses, confirming their essential roles in ligand binding and receptor activation (Fig. 1i, j; Supplementary information, Fig. S5 and Table S4). By integrating these structural findings with functional and expression data (Supplementary information, Table S5), we can elucidate the ligand recognition and binding mechanisms of SUCR1 in greater detail.

Comparisons of the above three complex structures reveal similar overall structural arrangements of SUCR1, with RMSD values for Cα atoms of the entire receptor ranging from 0.33 Å to 0.50 Å (Fig. 1k, l). Considering the similarity of the three complexes, we chose the succinic acid–SUCR1 complex for a detailed analysis of SUCR1 activation. Structural comparison of the active succinic acid–SUCR1 complex with the inactive rat SUCR1 (PDB: 6RNK)¹² reveals that the cytoplasmic end of TM6 in succinic acid-bound SUCR1 displays a pronounced outward movement while the cytoplasmic part of TM7 shifts inward. This rearrangement accommodates the C-terminal α5 helix of the Gα_i subunit, a key feature of class A GPCR activation (Fig. 1m). In general, many GPCRs are able to sense ligand binding via a conserved toggle switch, exemplified by W^6.48 of β₂AR. In the corresponding position of the toggle switch,¹³ SUCR1 has F245^6.48, which does not directly contact any of the agonists mentioned above. Instead, L102^3.32 and F285^7.43 directly interact with the alkyl portion of the ligands (Fig. 1n), pushing F285^7.43 downward to pack against L106^3.36 and F245^6.48, resulting in rotations of the P^5.50–I^3.40–F^6.44 motif. This chain of conformational changes ultimately leads to the outward bending of TM6 to accommodate G_i coupling.

In this study, we have determined cryo-EM structures of SUCR1 in complex with succinic acid, maleic acid, and compound 31. The detailed structural analysis provided not only enhances our understanding of SUCR1–ligand interactions, but also underscores the potential of structure-based drug design in developing targeted therapies for diseases associated with dysregulated succinic acid signaling, such as hypertension, inflammation, and metabolic disorders. The identification of key residues involved in ligand binding and receptor activation presents specific targets for the development of drugs aimed at modulating SUCR1 activity with high specificity and efficacy.

In conclusion, this study not only contributes to the expanding repository of GPCR structural biology, but also represents a pivotal step forward in the quest for targeted therapeutics. The comprehensive structural basis for SUCR1 activation provided herein highlights the receptor’s potential as a versatile and valuable target for drug design and discovery. As we continue to unravel the complex signaling networks mediated by SUCR1, the prospects for developing novel therapeutic strategies that harness the power of structure-based drug design become increasingly tangible, offering hope for the effective management and treatment of a wide range of diseases.

Author contributions

H.E.X. initiated the project. C.L. designed and screened the expression constructs of SUCR1, prepared protein samples of succinic acid–SUCR1–G_i complex, maleic acid–SUCR1–G_i complex, and compound 31–SUCR1–G_i complex for cryo-EM data collection, participated in cryo-EM grid inspection, and executed the functional studies. H.L. performed cryo-EM grid preparation, data acquisition, structure determination and model building, and prepared the draft of the manuscript and figures. J.L. performed cell-based functional assay and participated in figure preparation. X.H. performed molecular dynamics simulations to validate ligand binding pose in SCUR1. H.Z. synthesized the compound 31 for structural study under the guidance of W.F. H.E.X. wrote the manuscript with input from all authors.

Data availability

The cryo-EM density maps and corresponding atomic coordinates for the SUCR1–G_i complexes bound to succinic acid, maleic acid, and compound 31 have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB), respectively, with the following accession codes: EMD-39374 and 8YKW for the SUCR1–G_i complex bound to succinic acid; EMD-39375 and 8YKX for the SUCR1–G_i complex bound to maleic acid; EMD-39373 and 8YKV for the SUCR1–G_i complex bound to compound 31.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41422-024-00984-7.