Structures of ATP-binding cassette transporter ABCC1 reveal the molecular basis of cyclic dinucleotide cGAMP export

Abstract

Cyclic nucleotide GMP-AMP (cGAMP) plays a critical role in mediating the innate immune response through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. Recent studies showed that ATP-binding cassette subfamily C member 1 (ABCC1) is a cGAMP exporter. The exported cGAMP can be imported into uninfected cells to stimulate a STING-mediated innate immune response. However, the molecular basis of cGAMP export mediated by ABCC1 remains unclear. Here, we report the cryoelectron microscopy (cryo-EM) structures of human ABCC1 in a ligand-free state and a cGAMP-bound state. These structures reveal that ABCC1 forms a homodimer via its N-terminal transmembrane domain. The ligand-bound structure shows that cGAMP is recognized by a positively charged pocket. Mutagenesis and functional studies confirmed the roles of the ligand-binding pocket in cGAMP recognition and export. This study provides insights into the structure and function of ABCC1 as a cGAMP exporter and lays a foundation for future research targeting ABCC1 in infection and anti-cancer immunity.

Keywords: ABCC1/MRP1; cGAMP export; cGAS-STING pathway; innate immunity.

Figure 1.. In vivo and in vitro characterization of human ABCC1

(A) cGAMP quantification using ELISA from lysates and supernatants of HEK293T cells transfected by indicated plasmids. Intracellular and extracellular amounts of cGAMP were calculated before calculating the percentage of extracellular cGAMP. (B) IFN-β luciferase reporter assay using HEK293T cells transfected with indicated plasmids. Values are normalized to empty vectors only. (C) Size-exclusion chromatography (SEC) of hABCC1 reconstituted in the amphipol PMAL-C8. (D) SDS-PAGE analysis of fractions from SEC. (E) ATPase activity of dimeric and monomeric hABCC1 without and with 1 mM cGAMP. All ATPase activity values are normalized to dimeric hABCC1 without cGAMP. (F) ATPase activity of dimeric hABCC1. By nonlinear regression of the Michaelis-Menten equation, the K_M for ATP was determined to be 104.5 μM and the maximal ATPase activity was determined to be 16.8 nmol/mg/min (assuming a pre glycosylation molecular mass of 398.3 kDa, the specific ATP turnover rate is 6.7 min⁻¹). (G) ATPase activity of dimeric hABCC1 at different cGAMP concentrations. The protein and ATP concentrations were kept at 100 nM and 1.0 mM, respectively, for all measurements. Dashed lines represent the ATPase activity in the absence of cGAMP. The K_M value for cGAMP was determined to be 230 μM. (H) Representative live cell confocal images of HEK293 FT cells expressing ABCC1 1–10 and 11 split GFP constructs with Cell Mask staining in red and GFP in green. The colocalization of ABCC1-GFP and Cell Mask is represented in yellow. (I) Signal intensity of each channel along the white arrows in the composite images in H. For panels (A), (B), and (E), the data are mean ± s.e.m. with each dot representing a biological replicate (n = 3). Two-tailed Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant.

Figure 2.. Molecular architecture of apo hABCC1

(A) Cryo-EM density map of hABCC1 homodimer viewed along the membrane plane. The two promoters are colored salmon and purple, respectively. (B) Ribbon representations of the structural model of hABCC1 homodimer in (A). (C) Schematic of domain structure of hABCC1 containing TMD0, L₀ linker, and the pseudo-symmetric TMD1 and TMD2 coupled to NBD1 and NBD2, which form the exporter core. (D) Ribbon representation of a hABCC1 protomer. TMD0 is colored red, the L₀ linker magenta, TMD1 and NBD1 green, and TMD2 blue. The dashed line represents the region not observed in the cryo-EM map and not included in the model. Please also see Figure S1.

Figure 3.. The dimerization interface of hABCC1

(A) Cytoplasmic view of the transmembrane region of hABCC1 protomer represented in ribbon. TM bundles 1 and 2 forming the transporter core and TMD0 are shaded with different colors. (B) Extracellular view of hABCC1 dimer with cylinders representing α-helices. Transmembrane helices of TMD0 are colored red, the L₀ linker is colored pink, and the helices in TMD1 and TMD2 are colored green and blue, respectively. Each protomer is shaded with different colors and helices of one protomer are labeled with ′ to distinguish them from the other protomer. (C) Close-up view of the hABCC1 dimer interface with residues mediating dimerization are represented by sticks. The green stick model shows a cholesterol hemisuccinate molecule at the dimer interface. (D) Residues interacting with the cholesterol hemisuccinate (green) at the dimerization interface are represented by sticks. (E) cGAMP quantification using ELISA from lysates and supernatants of HEK293T cells transfected with indicated plasmids. (F) IFN-β luciferase reporter assay using HEK293T cells transfected with indicated plasmids. Values are normalized to empty vector only. (G) cGAMP quantification using ELISA from lysates and supernatants of Abcc1^−/− B16F10 cells expressing hABCC1 mutant. For (E), (F), and (G), the data are mean ± s.e.m. with each dot representing a biological replicate (n ≥ 3). Two-tailed Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant. Please also see Figure S1.

Figure 4.. Interdomain interactions within hABCC1

(A and B) Cryo-EM density maps and ribbon representations of apo-hABCC1 and bABCC1. Green ellipses show the densities for the loops mediating contacts between TMD0 and transporter core in hABCC1, while red ellipses show lack of such densities in bABCC1. (C) Ribbon representation of hABCC1 from within the plane of the membrane. TMD0 is colored salmon, the L₀ linker magenta, TMD1 green, and TMD2 blue. Interdomain interactions within hABCC1 molecule are outlined by the boxes and close-up views are shown in the indicated panels. (D) Close-up view of the interactions between the extracellular loop of TMD0 containing the disulfide bond and TMD1. Dashed line indicates hydrogen bond. (E) Close-up view of a loop of TMD0 interacting with the L₀ linker at the inner leaflet of the membrane. (F) Close-up view of interactions between the L₀ linker and TMD2. Please also see Figure S2.

Figure 5.. Cryo-EM structure of cGAMP bound hABCC1

(A) Overall structure of hABCC1 bound to cGAMP in an inward-facing conformation. The inset shows the location of the cGAMP binding site. The cGAMP molecule is shown by the magenta space-filling model. (B) Cryo-EM density maps of the two protomers of hABCC1 dimer purified in the presence of cGAMP. The cryo-EM density and ribbon representation of the hABCC1 protomer bound to cGAMP are colored blue. The cryo-EM density and ribbon representation of the hABCC1 protomer with no clear density at the substrate binding site are colored salmon. The maps are contoured at 36σ. Please also see Figures S2 and S3.

Figure 6.. Structure of the cGAMP binding pocket

(A) Close-up view of the cGAMP binding site from within the plane of the membrane. Side chains of the residues surrounding cGAMP are shown as sticks. Dashed lines indicate hydrogen bonds. The cryo-EM density is contoured at 12σ. (B) Schematic representation of cGAMP-hABCC1 interactions. Residues forming H-pocket are indicated in gray boxes, while residues forming P-pocket are indicated in cyan boxes. The dashed lines indicate hydrogen bonds. (C) The electrostatic surface of the cGAMP binding site of hABCC1. Scale: red, negative (5 kT/e); blue, positive (+5 kT/e). The side chains of residues surrounding cGAMP are shown as sticks. (D) Superposition of cGAMP-bound hABCC1 and LTC₄-bound bABCC1 structures. cGAMP and LTC₄ are represented by the orange and teal space-filling models, respectively. The inset shows a close-up view of cGAMP and LTC₄ in stick models. Phosphodiester linkage of cGAMP overlaps with GSH of LTC₄. (E) Superposition of apo-hABCC1 structure with cGAMP-bound hABCC1 structure. cGAMP is represented by the space-filling model. (F) Local conformational changes of hABCC1 upon cGAMP binding. Arrows indicate the movements of several key residues interacting with cGAMP. Please also see Figures S3, S5, S6, and S7.

Figure 7.. Mutations at the ligand binding pocket reduce cGAMP export

(A) Mutations of residues interacting with cGAMP affect extracellular cGAMP amounts. cGAMP quantification using ELISA from lysates and supernatants of HEK293T cells 13 h after transfection of indicated hABCC1 plasmids. (B) cGAMP quantification using ELISA from lysates and supernatants of Abcc1^−/− B16F10 cells expressing hABCC1 mutants. (C) ATPase activity fold induction of dimeric WT and mutant hABCC1 in the absence and presence of 1 mM cGAMP. All ATPase activity values are normalized to dimeric hABCC1 in the absence of cGAMP. (D) Mutations affecting cGAMP export also affect STING-mediated signaling in cells. IFN-β luciferase reporter assay using HEK293T cells transfected with indicated plasmids. Values are normalized to empty vector only. (E) ATPase activity fold induction of dimeric WT in the absence and presence of 1 mM 2′3′-cGAMP or 3′3′-cGAMP. All ATPase activity values are normalized to dimeric hABCC1 in the absence of cGAMP. (F) Mutation of catalytic glutamate residue (E1455) in the consensus site abolishes cGAMP export by hABCC1. cGAMP quantification using ELISA from lysates and supernatants of HEK293T cells trasfected by indicated hABCC1 plasmids. (G) Proposed mechanism of cGAMP export mediated by ABCC1. hABCC1 is shown by the ribbon representation, while cGAMP and ATP are represented by the space-filling model. For (A), (B), (C), (D), (E), and (F), the data are mean ± s.e.m. with each dot representing a biological replicate (n ≥ 3). Two-tailed Student’s t-test: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; NS, not significant. Please also see Figures S4, S5, S6, and S7.