The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation3,4,5. Because SUCNR1 senses succinate as an immunological danger signal6—which has relevance for diseases including ulcerative colitis, liver fibrosis7, diabetes and rheumatoid arthritis3,8—it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo.
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Structure factors and coordinates of the rat SUCNR1–Nanobody6 and the SUCNR1(K181.31E/K2697.32N)–Nanobody6–NF-56-EJ40 complex structures have been deposited in the Protein Data Bank (PDB) under accession codes 6IBB and 6RNK, respectively. All source data associated with the paper (in addition to those deposited) are provided as Supplementary Information.
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We thank C. Schleberger for help with implementing crystallographic data processing tools; T. Huber, R. Link and A. Winterhalter for help with nanobody generation; P. Loesle for biochemical assay support; S. Haenni and S. Holzinger for molecular biology support; and E. Loetscher for providing the rat SUCNR1-CHO cell line.
All authors are employees of Novartis Pharma AG.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Edward Chouchani, Mike Murphy, Janos Peti-Peterdi and Gebhard Schertler for their contribution to the peer review of this work.
Extended data figures and tables
Levels of the Krebs cycle intermediate succinate are increased under certain conditions such as hypoxia, necrosis, ischaemia reperfusion and inflammation. The ways in which succinate concentrations increase in the mitochondrion are shown in green. Mitochondrial reactive oxygen species result from the reversed electron transport (RET) chain driven by an increase in succinate. Succinate is transported into the cytoplasm, where it can stabilize HIF1α and increase the expression of genes that have HIF-responsive elements, such as IL1Β. Further succinate is exported into the local extracellular environment, where it accumulates and binds and activates SUCNR1. Several of the consequences of this are shown in red. GABA, γ-aminobutyric acid; HIF, hypoxic inducible factor; IL-1, interleukin-1; mROS, mitochondrial reactive oxygen species; SDH, succinate dehydrogenase.
Extended Data Fig. 2 Interaction of Nanobody6 with rat and human SUCNR1 and characterization of the effects of glycerol and 2,5-hexanediol on rat SUCNR1.
a, b, Nanobody6 increases the thermal stability of rat SUCNR1 in nano-DSF thermal shift assays. The average melting curves in the absence (a) and presence (b) of Nanobody6 are shown (n = 3; technical replicates). The experiment was repeated independently 3 times with similar results. c, Analytical size-exclusion chromatography shows a clear shift in the peak of the rat SUCNR1–Nanobody6 complex compared to the peak of the receptor alone. The complex samples contain a 1.2 molar excess of Nanobody6 over receptor. One of n = 2 independent experiments is shown. d, [35S]GTPγS assay on wild-type rat SUCNR1 in the absence or the presence of increasing concentrations of Nanobody6. The average curves of n = 3 independent experiments are shown; data are mean ± s.d. Average half-maximum effective concentration (EC50) values from n = 3 independent experiments are listed; data are mean ± s.d. e, Analytical size-exclusion chromatography shows a clear shift in the peak of the N-terminal BRIL-fused human SUCNR1–Nanobody6 complex compared to the peak of the receptor alone. One of n = 2 independent experiments is shown. f, [35S]GTPγS assay on wild-type human SUCNR1 in the absence or the presence of increasing concentrations of Nanobody6. The average curves of n = 3 independent experiments are shown and average EC50 values from n = 3 independent experiments are listed; data are mean ± s.d. g, Analytical size-exclusion chromatography of rat SUCNR1 purified in the absence or the presence of 10% glycerol. One of n = 2 independent experiments is shown. h, Glycerol increases the thermal stability of rat SUCNR1 as evidenced from nano-DSF assays (n = 3 technical replicates; bars represent mean values; individual data points are indicated by circles). The control sample was purified in the presence of 10% glycerol. All other samples contain rat SUCNR1 purified without glycerol, to which the respective final glycerol concentration was added. The experiment was repeated independently twice with similar results. i, 2,5-Hexanediol decreases the thermal stability of rat SUCNR1 in nano-DSF assays (n = 3 technical replicates; bars represent mean values; individual data points are indicated by circles). The experiment was repeated independently twice with similar results. j, k, [35S]GTPγS assay of rat SUCNR1 (j) and human S1P1R (k) in the absence or the presence of 1% (109 mM) glycerol. The average curve of n = 3 independent experiments and the individual data points of each experiment are shown. Data are mean ± s.d. Source Data
Extended Data Fig. 3 Purification, crystallization and electron-density map quality of the rat SUCNR1–Nanobody6 complex, with detailed binding modes of glycerol and 2,5-hexanediol.
a, Analytical size-exclusion chromatography and SDS–PAGE analysis of crystallization samples of the rat SUCNR1–Nanobody6 complex. Shown is a representative experiment of n = 5 independent experiments with similar results. For gel source data, see Supplementary Fig. 1a. b, Initial crystallization hits for the rat SUCNR1–Nanobody6 complex (top) and optimized crystals used for data collection (bottom). Shown are representative experiments of n = 20 independent experiments. c, The 2Fo − Fc electron density map contoured at 1.5σ for a part of Nanobody6. d, The 2Fo − Fc electron-density map contoured at 1.5σ for helix VII in rat SUCNR1. e, f, Fo − Fc composite omit map for glycerol (e) and corresponding 2Fo − Fc electron-density map after refinement (f). Both maps are contoured at 1.5σ. g, h, Fo − Fc composite omit map for 2,5-hexanediol (g) and corresponding 2Fo − Fc electron-density map after refinement (h). Both maps are contoured at 1.5σ. i, j, Detailed views of the side-chain environment around 2,5-hexanediol (i) and glycerol (j). Hydrogen bonds are indicated by dashed lines. For clarity, only residues within a distance of 4 Å are shown. Source Data
Extended Data Fig. 4 Rat SUCNR1 adopts an inactive conformation in complex with Nanobody6, which binds to the intracellular side via an extended CDR3 loop.
a, Structural alignment of helix VI in rat SUCNR1 and the active and inactive states of β2-AR, with the positions of key residues as hallmarks for inactive and active receptor states. SUCNR1 is shown in blue, inactive β2-AR in red and active β2-AR in pink. Alignment of helix VI (left) and side-chain positions of key residues (R3.50 and Y7.35) (right) indicate an inactive state for rat SUCNR1. b, Structural alignment of helix VI and key residues (R3.50 and Y7.35) of rat SUCNR1 (blue) and the P2Y1 receptor (orange) in the inactive conformation. c, Superposition of the rat SUCNR1–Nanobody6 complex with the GαS subunit from the β2-AR GS-protein trimer structure (PDB ID: 3SN6). Rat SUCNR1 is shown in blue, Nanobody6 in orange and the GαS subunit in red. The G-protein and the Nanobody6-binding site partially overlap. d, Magnified view of the overlap between the G-protein and the Nanobody6-binding site of rat SUCNR1. e, Structural alignment of nanobodies used to crystallize GPCRs. The extended CDR3 of Nanobody6 forms a helical secondary structure. f, Sequence alignment of the GPCR-stabilizing nanobodies shown in e. The PDB IDs of the respective GPCR–nanobody complex structures are listed and the CDR3 region is highlighted by a black bar.
a, The helical structure elements as observed in the crystal structure of apo rat SUCNR1 are indicated. Sequences corresponding to ECL1 and ECL2 are boxed in blue and the non-conserved residues K181.31 and K2697.32 are marked by blue arrows. Yellow arrowheads indicate residues that were previously reported to be involved in succinate-induced receptor activation. Green dots indicate residues that are involved in NF-56-EJ40 binding in the humanized rat SUCNR1 structure. b, The sequence alignment of SUCNR1 from various species is colour-coded from turquoise (variable) to dark pink (conserved), similar to the colours used in Extended Data Fig. 6a, b, on the basis of analysis with ConSurf. Residues involved in the binding of Nanobody6 are indicated by blue triangles.
a, Side view (top) and top view (bottom) of the hydrophobic pocket located below the glycerol molecule. The sequence conservation within the SUCNR1 receptor family is indicated by colour, ranging from turquoise for highly variable residues to dark pink for highly conserved residues. The hydrophobic pocket is shown as a surface, colour-coded by charge. The glycerol molecule is shown as yellow sticks. b, The same orientations as in a are shown. Residues forming the deep hydrophobic pocket are shown as sticks, colour-coded by sequence conservation as in a. Residues that were previously reported to be involved in succinate-induced receptor activation are coloured yellow. c, Residues in the environment of ECL2 are shown as sticks and hydrogen bonds are shown as dashed black lines. Residues that have previously been reported to have an effect on succinate binding by the receptor are shown in yellow2; R2516.58, which was identified in a second study15, is shown in green.
Extended Data Fig. 7 Identification of critical residues that impart species selectivity for antagonist binding.
a, Side view (left) and top view (right) of the potential binding mode of the antagonist NF-56-EJ40 (shown in pink), obtained by molecular modelling based on the crystal structure of apo rat SUCNR1. Residues within 4 Å of NF-56-EJ40 are shown as sticks. Red arrows point towards two sites in which potential steric clashes may occur with rat-SUCNR1-specific residues K181.31 and K2697.32, which are shown in blue. For clarity, ECL2, helix IV and helix V are omitted in the side view. b, Radioligand competition binding experiments with unlabelled NF-56-EJ40 on human SUCNR1 mutant proteins. Curves were calculated from n = 3 independent experiments; data are mean ± s.d. Individual data points are shown. Source Data
Extended Data Fig. 8 Purification, crystallization and electron-density map quality of the rat SUCNR1–Nanobody6–NF-56-EJ40 complex and structural changes in SUCNR1 after antagonist binding.
a, Analytical size-exclusion chromatography and SDS–PAGE analysis of crystallization samples of the humanized rat SUCNR1–Nanobody6–NF-56-EJ40 complex. Shown is a typical result from n = 2 independent experiments. Although partial complex aggregation was observed, this did not interfere with crystallization. For gel source data, see Supplementary Fig. 1b. b, Top, initial crystallization hits for the humanized rat SUCNR1–Nanobody6–NF-56-EJ40 complex, shown in normal (left) and cross-polarization (right) imaging modes. Bottom, optimized crystals used for data collection shown in normal (left) or cross-polarization (right) imaging modes. A typical result from n = 3 independent experiments is shown. c, The 2Fo − Fc electron density map contoured at 1.5σ for a part of Nanobody6. d, The 2Fo − Fc electron density map contoured at 1.5σ for helix VII in humanized rat SUCNR1. e, Fo − Fc composite omit map (top) for NF-56-EJ40 and glycerol contoured at 1.5σ is shown in orange. The 2Fo − Fc map (bottom) for NF-56-EJ40 and glycerol after refinement contoured at 1.5σ is shown in blue. f, Top view of apo rat SUCNR1 (shown in orange) and humanized rat SUCNR1 (shown in blue) in complex with NF-56-EJ40 (shown as green sticks). Large structural rearrangements are indicated by red arrows. Note that ECL2 is completely structured in the humanized rat SUCNR1 structure. g, Top view of the NF-56-EJ40-binding site in humanized rat SUCNR1 overlaid with apo rat SUCNR1. Important side chains around NF-56-EJ40 (shown in green) are shown as sticks and are coloured blue for humanized rat SUCNR1 or orange for apo wild-type rat SUCNR1. For clarity, only the backbone of the humanized rat SUCNR1 is shown in cartoon representation. h, Side chains that directly interact with NF-56-EJ40 via hydrogen bonding, π–π stacking and cation–π stacking are listed in black. The hydrogen-bonding interactions are shown by black dashed lines and the π–π and cation–π interactions are shown by green dashed lines. Additional residues with van der Waals interactions are listed in green, and their interaction surfaces are indicated by solid green lines. i, Top view of the humanized rat SUCNR1 (blue) in complex with NF-56-EJ40 (green sticks) and of the apo rat SUCNR1-derived model of human SUCNR1 (red) with the binding mode of NF-56-EJ40 (pink) from molecular-docking studies, for which details are shown in Extended Data Fig. 7a. Note how both NF-56-EJ40 poses differ considerably, as indicated by red arrows. j, Detailed views of the NF-56-EJ40-binding site. For clarity, only side chains are shown. Humanized rat SUCNR1 is shown in blue; wild-type rat SUCNR1 is shown in orange. Note the side-chain flips for R953.29, L983.32, H993.33 and Y171 between both structures. k, Top view of wild-type rat SUCNR1 structure (left), the apo rat SUCNR1-based homology model of human SUCNR1 in complex with NF-56-EJ40 (middle) and the humanized rat SUCNR1 structure in complex with NF-56-EJ40 (right). The surface is shown coloured by electrostatic charge. The two key positions (K/E1.31 and K/N7.32) are highlighted by arrows. NF-56-EJ40 is shown in yellow as a ball-and-stick model. Note the differences between the NF-56-EJ40-binding mode determined in the modelled structure and in the crystal structure, and between the surface charge distributions in rat, human and humanized rat SUCNR1. Source Data
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Haffke, M., Fehlmann, D., Rummel, G. et al. Structural basis of species-selective antagonist binding to the succinate receptor. Nature 574, 581–585 (2019) doi:10.1038/s41586-019-1663-8