. Author manuscript; available in PMC: 2025 Feb 13.

Published in final edited form as: Trends Immunol. 2023 May 4;44(6):450–467. doi: 10.1016/j.it.2023.04.006

Structure-based mechanisms of 2′3′-cGAMP intercellular transport in the cGAS-STING immune pathway

PMCID: PMC11824902 NIHMSID: NIHMS2011557 PMID: 37147228

Abstract

Upon activation by dsDNA, the cytosolic dsDNA sensor cGAS synthesizes the diffusible cyclic dinucleotide 2′3′-cGAMP, which subsequently binds to the adaptor STING, triggering a cascade of events leading to an inflammatory response. Recent studies have highlighted 2′3′-cGAMP’s role as an ‘immunotransmitter’ between cells, a process facilitated by gap junctions, as well as specialized membrane-spanning importer and exporter channels. This review highlights recent advances from a structural perspective of intercellular trafficking of 2’3’-cGAMP, with particular emphasis on importer SLC19A1 binding to 2’3’-cGAMP, as well as the significance of associated folate nutrients and antifolate therapeutics. This information provides a path forward for a structure-guided understanding of the transport cycle in immunology, as well as candidate targeting approaches towards therapeutic intervention in inflammation.

Signaling molecule 2′3′-cGAMP functions as both a second messenger and an immunotransmitter

The cGAS-STING innate immune pathway represents a host defense mechanism in metazoans to limit the harmful effects of invading organisms [1,2]. The primary function of this pathway is to detect and respond to the presence of foreign double-strand DNA (dsDNA) in the cytoplasm [3–12]. Structural and biochemical studies have established that activation of cGAS by dsDNA is triggered by the formation of multivalent protein-DNA interactions [13–17], as well as the formation of liquid-like condensates [17,18], with DNA sensing of cGAS occurring in a DNA length- and concentration-dependent manner [19,20]. However, given the non-sequence-specific binding of DNA to cGAS [21,22], this pathway can also generate an inflammatory response to mislocalized self-nucleic acids [6,23]. cGAS adopts a common cGAS/DncV-like nucleotidyltransferase (CD-NTase) fold [24] to catalytically generate 2′3′-cGAMP from ATP and GTP upon dsDNA binding [13,14,25–27]. The diffusible 2′3′-cGAMP subsequently activates the adaptor protein STING [28–30], with structural studies identifying conformational changes in STING’s cytoplasmic and transmembrane domains on complex formation [10,12,31,32]. Upon activation, STING translocates from the endoplasmic reticulum (ER) to the Golgi apparatus to be oligomerized or polymerized [33]. Activation of STING triggers phosphorylation of downstream kinases and transcription factors [34], leading to the upregulation of cytokine and type I interferon (IFN-Is) production [7,35]; this constitutes a crucial step in the innate immune response to viral and bacterial infections. Chronic activation of cGAS-STING by autologous DNA underlies triggering of rare inflammatory diseases such as Aicardi-Goutières syndrome [30,36,37] and contributes to other disease processes related to autoimmunity, neurodegeneration, aging, and cancer [30,35,38–42].

Cyclic dinucleotides (CDNs), which are small molecules composed of two nucleotides joined by phosphodiester bonds to form a ring structure, include cyclic di-AMP (cAA), cyclic di-GMP), and cyclic GMP-AMP (cGAMP). CDNs with 3’-5’ linkages were originally discovered to be signaling molecules in bacteria and found to play a key role in bacterial anti-phage response, virulence, motility, metabolism, and survival [43–47]. Although CDNs with 3’-5’ linkages can also activate STING, 2′3′-cGAMP (with a 2’-5’ linkage at the GpA step) binds to STING with a significantly higher affinity [27,48]. One advantage of CDN second messenger signals is that they allow amplification and transmission of the input signal. Notably, recent studies identified 2′3′-cGAMP as an ‘immunotransmitter’ between cells [49–52], in addition to its role as the second messenger signaling molecule in cells. Central to this idea is the observation that 2′3′-cGAMP can exit from producing cells and enter into responding cells (shown schematically in Figure 1). Studies in tumor models and immunotherapy have shown that extracellular 2′3′-cGAMP promotes the production of inflammatory cytokines (including IFNs), facilitates immune cell activation, modulates local antitumor immunity, and affects tumor metastasis [49,53]. In turn, the extracellular membrane-bound protein ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) degrades extracellular 2′3′-cGAMP to attenuate cGAS-STING signaling [54].

Figure 1. — Overview of the intercellular transport of 2’3’-cGAMP. 2’3’-cGAMP binds to STING dimer located in the endoplasmic reticulum (ER). STING activation induces multiple cellular functions, such as cytokine induction. Gap junctions and human host defense peptides can mediate 2′3′-cGAMP transfer to neighboring cells. Extracellular 2′3′-cGAMP can be imported into cells *via* various channels, including SLC19A1 in U937 and THP-1 cells, ATP-activated P2X7R receptors, and VRACs involved in bi-directional 2′3′-cGAMP transfer. MRP1, an ATP-binding cassette (ABC) exporter, is the newly identified exporter of 2′3′-cGAMP. Finally, extracellular 2′3′-cGAMP levels can be attenuated by membrane-bound glycoprotein ENPP1.

Like other CDNs, 2′3′-cGAMP is negatively charged and cannot passively cross the cell membrane to enter or exit cells due to both its charge, shape and size. Therefore, transport proteins are needed to shuttle CDNs across the membrane (Figure 1). These transport proteins can adopt either passive or active mechanisms of transport. Passive transporters move substrates based on electrochemical gradients, while active transporters use the energy of ATP hydrolysis to move substrates across membranes, sometimes against a concentration gradient. The need for transport proteins in CDN transport is important for cell-specific cellular signaling and immune response pathways that rely on the efficient movement of these immune signaling molecules across membranes. Such transport could be mediated by gap junctions and specific host defense peptide scaffolds on the one hand and importers and exporters on the other (Figure 1). Given the size, shape, charge and chemical properties of CDNs, there are questions on whether CDNs are transported as monomers or dimers, as well as the oligomerization status and conformational (inward-open, occluded and outward-open) states of the transporters during the transport cycle. Given the diversity of CDN immunotransmitters, and the electrostatics of individual transport channels, there are challenges regarding how transport of CDNs is coupled to countertransport of ions in the opposite direction.

Previous reviews have extensively covered the structural biology of the cGAS-STING surveillance pathway and the crucial role of 2′3′-cGAMP as a second messenger within cells [10,12,24,32,55]. In this review, we summarize recent advances in the transport and regulation of 2′3′-cGAMP-mediated intercellular cGAS-STING signaling from a structural perspective, providing a deeper and more precise understanding of its role and emerging insights into underlying mechanisms. As featured in Figure 1, we discuss structural features of gap junctions and human host defense peptides [56,57], topologies known to traffic 2′3′-cGAMP between cells. In addition, we discuss the structural and functional features of the importers and exporters of extracellular 2′3′-cGAMP [50,51,58–61], with an emphasis on SLC19A1, for which structures have been solved with bound CDNs, folates, and anti-folates. The challenge has been to explore and develop a molecular understanding of cell type specificity and expression levels of 2’3’-cGAMP immunotransporters and their species selectivity, as well as the efficiency, selectivity, and directionality of 2’3’-cGAMP transport.

These advances may pave the way for structure-guided therapeutic interventions, offering new opportunities for developing effective immunotherapeutic strategies against cancer and other diseases.

TRAFFICKING OF 2′3′-cGAMP BETWEEN CELLS

We outline below two examples of intercellular trafficking of 2’3’-cGAMP, namely by gap junctions and by human host defense peptide LL-37.

Intercellular 2’3’-cGAMP transport mediated by gap junctions

Gap junctions are specialized intercellular channels that allow for direct communication and exchange of small molecules between adjacent cells. Intercellular transport of 2′3′-cGAMP was first identified in virus-infected cells, where 2′3′-cGAMP traffics between cells via gap junctions formed by clusters of connexin proteins [62,63]. 2’3’-cGAMP transfer mediated by gap junctions can occur between cells of the same or different types and is not impacted by 2’3’-cGAMP-specific ENPP1 nuclease. Humans express 21 connexin isoforms in a cell-type-specific fashion [64–66]. Reports confirmed that connexins 43 and 45 are required for 2′3′-cGAMP translocation to adjacent cells, but other connexin proteins may also be involved [67–69]. Notably, 2′3′-cGAMP translocation from virus-infected endothelial cells to macrophages via connexin 43 enhances the antiviral response [68], suggesting that this mechanism promotes antiviral immunity. The connexin-mediated 2′3′-cGAMP transport has been reported to regulate inflammatory responses in cancer, alcohol-induced hepatocyte injury, and inflamed joints [68–70].

Structural studies of connexins mediating gap junctions outlined in Box 1 and Figure 2 have identified and defined connexin oligomerization patterns leading to channel generation with its inner pores displaying distinct electrostatics and constriction segments regulating ligand transport between cells [71–73]. Gap junctions allow for direct exchange of small molecules between adjacent cells, while hemichannels are similar structures that are present on cell surfaces and can release or take up small molecules into or out of the cell. It is possible that these different types of channels may play a role in the transport of CDNs between cells, but further research will be needed to determine their specific functions in this context.

Box 1. Structural biology of gap junctions.

The crystal structures of gap junctions comprising connexin 26 [72] and cryo-EM structures of connexins 46 and 50 [71] involved in forming lens gap junction channels revealed that gap junctions share a plaque-like large architecture by assembling twelve connexin subunits that form intercellular channels connecting neighboring cells [73] (Figure 2). Within the plasma membrane, six connexins are organized into a hemichannel topology, with hemichannels from neighboring cells docking together to build up complete cell-to-cell channels. Each connexin contains a typical TM (transmembrane) four-helix bundle, two extracellular loops E1 and E2, a cytoplasmic loop, an N-terminal helix (NTH), and a C-terminal segment (CT) (Figure 2).

Comparison between the structures of different gap junctions reveals distinct electrostatic pore pathways with shared regions of charge potential and steric constriction sites formed by NTH domains that narrow the cytoplasmic vestibule at both ends of the channel to approximately 10–12 Å [71]. The pore size (approx. 14 Å) is within the range defined by other linker protein channels and consistent with the general ability of gap junctions to allow the passage of mixed chemical messages between cells, including ions, metabolites, hormones [64,70], 2′3′-cGAMP and other molecules smaller than 1 kDa in size. Recent cryo-EM studies at high resolution have demonstrated a lipid-induced stabilization of the gap junction channel [62].

Figure 2. — Overview of the structural features of connexins (CXs) to form gap junctions [71–73]. The folding of connexins is shown in a ribbon representation based on the crystal structure of the human CX26 (PDB code: 2ZW3) and cryo-EM structure of the human CX46 (PDB code: 6MHQ) gap junctions. The hexametric CX26s in the open state have hemichannel activity, and the intercellular communication results from assembling twelve connexin subunits to connect neighboring cells.

Future efforts also need to be directed towards solving the structure of 2’3’-cGAMP-bound to the oligomeric state of connexins towards an understanding of the principles underlying transport of CDNs by gap junctions. Such an effort could guide the design of 2’3’-cGAMP binding pocket mutations, and in the case of connexin co-assembly in native lens gap junction channels, their impact on hereditary cataract formation.

2’3’-cGAMP intercellular transport mediated by human host defense peptide LL-37

Host human defense peptide LL-37 is a widely distributed peptide reported to possess multiple biological activities, including antibacterial, antiviral, and anti-inflammatory properties, and to promote wound healing [74]. A recent study reported that the combination of synthesized LL-37 with extracellular 2′3′-cGAMP could lead to efficient activation of STING immune signaling and enhancement of interferon production in THP-1 monocyte cells [75]. Additionally, LL-37 inducers, such as vitamin D3 and sodium butyrate, enhanced 2′3′-cGAMP-mediated immune responses by upregulating endogenous LL-37 expression in THP1-, Caco2-, iBMDM-, and RAW-Lucia ISG cells stably expressing interferon-stimulated response element (ISRE) luciferase reporter. Interestingly, this study also showed that LL-37 especially bound 2′3′-cGAMP and transferred 2′3′-cGAMP across the cell membrane in HEK293T and RAW264.7 cells.

Structural studies on host defense peptides LL-37 outlined in Box 2 and Figure 3 have led to proposed models ranging from tetrameric channels [76] to hexameric fibrils [77] facilitating intracellular transport. Consistently, in vitro assays showed that 2′3′-cGAMP-induced LL-37 oligomerization, with 2′3′-cGAMP observed to bind to soluble LL-37 and form precipitated aggregates [75]. These results suggest that LL-37 is a potential 2′3′-cGAMP carrier that might exhibit promising therapeutic possibilities in host immune regulation, though there is a need for further investigation of this system.

Box 2. Structural biology of human host defense LL-37 peptides.

NMR studies of LL-37 peptide in sodium dodecyl sulfate (SDS) or dodecyl phosphocholine (DPC) micelles revealed its helix-break-helix topology in the monomeric state, with unstructured N- and C-terminal ends [101]. By contrast, the crystal structure of LL-37 showed an anti-parallel dimer formed by two α-helices without supercoiling (Figure 3A) [102]. Three hydrophilic residues (Ser9, Lys12 and Glu16) form hydrophilic contacts at the dimer interface, which together with an extended hydrophobic core, contributes to high structural stability. The LL-37 peptide dimer carries a surface with extensive positive charges, potentially interacting with liposaccharide (LPS) or lipoteichoic acid (LTA) of the bacterial cell wall [103], negatively charged cell membrane lipids, as well as DNA, in vivo. Crystal structures were also determined of LL-37 complexed with the detergents DPC and lauryldimethylamine oxide (LDAO), with the binding of detergent molecules causing the structural transition of the N-terminus of LL-37 into a random coil conformation. This transition led to the exposure of Phe5 and Phe6 for hydrophobic interactions with the detergent alkyl chain. In addition, biochemical and biophysical studies demonstrated oligomerization of LL-37 under increased salt and at higher peptide concentrations [103].

A recent crystallographic structural study established that LL-37 forms a narrow tetrameric channel with a strongly charged core in the presence of detergents (Figure 3B) [76], with a chloride anion trapped between two arginine residues in the interior of the channel.

Another recent structural study determined the self-assembly of the human LL-37 active core (residues 17–29) into a protein fibril of densely packed helices (Figure 3C) [77]. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-37 (17–29) correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells.

Given the various multimeric topologies proposed for LL-37 peptide (Box 2 and Figure 3), it remains somewhat unclear how 2’3’-cGAMP is trafficked by LL-37. Insights into transport of 2′3′-cGAMP within a LL-37 channel/fibril topology will require structural characterization of 2′3′-cGAMP-bound LL-37 channel/fibril complexes. Such an effort, if successful, should address not only the oligomeric state of LL-37 peptide, but also define intermolecular contacts between 2’3’-cGAMP and LL-37 peptide.

IMPORTERS OF EXTRACELLULAR 2’3’-cGAMP

In addition to transmission through intercellular channels, extracellular 2’3’-cGAMP can enter the cytoplasm of targeting cells via specialized cell-surface importers that can handle its negative charge. Several distinct transmembrane importers have been identified – these include SLC19A1 and SLC46A2, as well as LRRC8 and P2X7R, with transport based on diffusion along physicochemical gradients.

SLC family importers

SLC19A1 is a folate-organic phosphate antiporter involved in the transport of folates and a variety of organic phosphates, such as thiamine derivatives and nucleotides, thereby representing the major transporter for both folate nutrients [78] and antifolate therapeutics [78–81]. SLC19A1 can transport various CDNs ranging from 2’3’-cGAMP and its thiophosphate analog, to bacterial and synthetic 2’3’ and 3’3’-linked CDN analogs, primarily in monocyte-derived cell lines with specificity for humans, while inactive in mice. To comprehensively identify potential importers of CDNs, two studies performed genome-wide CRISPR-Cas9 screens in the human monocytic THP-1 cells and monocyte-derived U937 cells, which were transduced with reporters to visualize STING activation upon CDN induction. These studies provided compelling evidence for the essential role of SLC19A1 in importing extracellular 2’3’-cGAMP and other CDNs [50,51].

Structural studies undertaken on the SLC19A1 importer (Figure 4A) and its complexes with CDNs (Figure 4B), folates, and antifolates (Figure 4C) outlined in Box 3, have identified the topology and electrostatics of the channel, the compact dual-molecule unit adopted by bound CDNs, as well as details of folate/antifolate ligand binding sites and intermolecular contacts stabilizing complex formation [78]. The structures of SCL19A1 reveal an overall positively charged interior cavity and cytosolic entrance, such that uptake of extracellular 2’3’-cGAMP must be coupled with counter transport of organic anions down a concentration gradient, thereby providing a driving force for substrate uptake by maintaining an appropriate anion concentration gradient. By analyzing these structures, insights have been gained into how SLC19A1 interacts with its substrates and how specific pore-lining mutations in the protein may affect its function. Specifically, SLC19A1 accommodates multiple CDNs with different compositions using a broad substrate recognition mechanism that incorporates conformational adjustments spanning a range of stacking, hydrogen bonding and charged intermolecular interactions. Notably, single point mutations on the CDN-binding pocket result in reductions in IRF3 phosphorylation, thereby identifying key residues involved in CDN transport. Thus, the structures and identification of key pore-forming residues provide a platform for a mechanistic understanding of loss-of-activity and disease-related mutants [78].

Box 3. Structural biology of SLC family importers bound to CDNs, folates and antifolates.

Cryo-EM structures have been solved of human SLC19A1 (Figure 4A) in the apo state and bound to 2’3’-cGAMP (Figure 4B), 2’3’-cGAMP^S (phosphate linkage replaced by thiophosphate), 2’3’-cAA and 3′3′-cAA, as well as a predominant natural folate 5-MTHF and a new-generation antifolate drug pemetrexed (PMX), through the utilization of a nanodisc-reconstituted SLC19A1-Fab system [78]. These results support SLC19A1 being a universal CDN second messenger importer, with the transport channel accommodating a diversity of CDNs with base and linkage diversity. The 12 TM (transmembrane) segments of SLC19A1 adopt a canonical major facilitator superfamily fold composed of N-terminal (TM1-TM6) and C-terminal (TM7-TM12) segments that together adopt an inward-open conformation (Figure 4B). Surprisingly, two molecules of CDNs are tightly intertwined and bound in the inward open cavity, closer to the intracellular side, as shown for the 2′3′-cGAMP bound complex [78]. Two bound CDNs pair with each other by extensive hydrogen bonding networks, as well as stacking interactions, resulting in a unique compact dual-molecule symmetric topological unit (Figure 4B). The authors also studied the impact of key single binding pocket residue alanine mutants of SLC19A1 on interferon response production, thereby identifying key residues involved in CDN recognition.

Structural and mutagenesis results demonstrate that SLC19A1 utilizes different binding pockets to recognize CDN- and folate-type substrates. The two CDN molecules bind within the SLC19A1 cavity as a compact dual-molecule unit (insert, Figure 4B) [78], while 5-MTHF [78], next-generation antifolate PMX [78] and chemotherapeutic agent methotrexate (MTX) [80] bind as monomers and are positioned at the same site within a negatively-charged pocket located at the upper and narrower segment of the channel (Figure 4C). Importantly, the pterin chromophores of the folates are positioned in an upright conformation in the context of an inward-open conformation. Notably, the dual-molecule CDNs are unable to bind in the narrower folate pocket, even as monomers.

Structural information is also available for the related proton-coupled folate transporter SLC46A1 (also called PCFT) (Figure 4D) both in the apo state and bound to PMX [79] (Figure 4E). Notably, in contrast to anion exchanger SLC19A1 that adopts an inward-open conformation in the apo- as well as CDN- (Figure 4B) and folate/antifolate-bound states (Figure 4C) [78,80], the cation exchanger SLC46A1 adopts an outward-open conformation in the apo- and antifolate-bound states (Figure 4E) [79]. The bound PMX is positioned in the middle of the channel and anchored in place through hydrogen bonding, hydrophobic and electrostatic contacts.

Given the larger volume of dimeric CDNs compared to monomeric folate/antifolate ligands, the dimeric CDNs cannot be accommodated in the narrow folate/antifolate binding pocket. The substrate binding pocket also exhibits selectivity, given that the methyl group (missing in folic acid) of antifolate 5-methyltetrahydrofolate (5-MTHF) is positioned in a hydrophobic environment [80]. Notably, given that CDNs and folates/antifolates bind in different pockets within the same substrate channel for transport, mutations in the CDN pocket were found to have an impact on the folate/antifolate pocket. A future challenge will be to extend the structural studies of the complex in the inward-open state to trap and structurally characterize CDNs bound to SLC19A1 in the outward-open and occluded states, thereby providing a fuller picture of the transport cycle.

SLC46A2 has been identified as the major 2′3′-cGAMP importer in human CD14⁺ monocytes and monocyte-derived macrophages [58]. This study also showed that related family members SLC46A1 and SLC46A3 could also import 2′3′-cGAMP. Notably, SLC46A family members are cation exchangers in contrast to SLC19A family members being anion exchangers.

Structural studies undertaken on the SLC46A1 importer (Figure 4D) and its complex with antifolate pemetrexed [79] outlined in Box 3 and Figure 4E, establish that the antifolate-bound SLC46A1 complex adopts an outward-open conformation, that contrasts with the inward open conformation observed for the antifolate-SLC19A1 complex (Figure 4C) [78].

The impact of structure-guided site-specific mutations has provided insights into mechanisms of proton coupled folate transport at acidic pH and towards the rational design of future generation antifolate drugs [79]. Thus, Arg, Asp, and Glu residues involved in missense mutations impacting on hereditary folate malabsorption have been shown to reposition during conformational transitions required for transport. Similarly, the structural information has indicated that the bound antifolate must engage both salt bridges at either end of the binding site to open an intracellular gate, thus highlighting the minimal length of substrates for optimal recognition impacting on drug design [79].

Once structures of 2’3’-cGAMP bound to SLC46A family importers becomes available, a comparative structural analysis should address whether a common mechanism of CDN import is mediated by SLC19A1 and SLC46A2 channels.

LRRC8 VRAC importers

Volume-regulated anion channels (VRACs) composed of a hexameric alignment of LLRC8 membrane-spanning proteins are activated in response to the hypotonic stress of low cellular osmolarity [82,83]. Based on a whole-genome CRISPR knockout screen in U937 Cas9-SLC19A1^−/− cells [61] and a screen of an anion channel inhibitors library on a cell-based viral infection platform [59], Leucine-rich repeat-containing protein 8A (LRRC8A) heteromeric channel, in complex with LRRC8C or LRRC8E, has been shown to be the widely expressed direct, bi-directional transporter of 2′3′-cGAMP and other CDNs [59,61]. By contrast, complexes containing LRRC8D inhibit the transport of 2′3′-cGAMP [61]. LRRC8 channels transport distinct anionic substrates across the plasma membrane in a range of cell types including fibroblasts and macrophages. This importer forms hexameric channels involving an obligatory LRRC8 subunit together with five LRRC8 paralogs. The heptameric LLRC8 channel can act bi-directionally; namely, it ‘passively’ mediates both the import and export of 2′3′-cGAMP, relying on the concentration gradient of CDN. In addition, LLRC8-mediated 2′3′-cGAMP transport can boost the antiviral interferon response in various primary cells such as murine embryonic fibroblasts (MEFs) [59], supporting the importance of transport of 2′3′-cGAMP by LLRC8 in the host defense against viral infection.

Structural studies undertaken on the LRRC8A importer outlined in Box 4 and Figure 5A establish that LLRC8A adopts a hexameric alignment that generates a transmembrane pore whose basic electrostatics and selectivity filter govern anion and 2′3′-cGAMP transport [84]. The transmembrane domain is structurally related to connexin proteins and contains a constriction within the channel (Figure 5B) lined by basic residues that acts as a selectivity filter involved in transport of anions by electrostatic interactions.

Box 4. Structural biology of VRAC LRRC8 importers.

Structures of homomeric channels of the obligatory human and mouse subunits LRRC8A or LRRC8D have been determined by cryo-EM (Figure 5A) [84,104–106]. LRRC8A coassembles to form a homohexameric channel with each monomer consisting of an extracellular cap, four transmembrane spanning helices TM1–4, a linker region, and a cytoplasmic leucine-rich repeat (LRR) domain on proceeding from the extracellular to the intracellular side. Its modular structure consists of a transmembrane pore with an excess of basic residues both in the extracellular selectivity filter and throughout the pore, thereby recruiting anions by electrostatic interaction. The transmembrane domain is wider towards the cytoplasm but constricted on the outside by a structural element which acts as a selectivity filter (Figure 5B) [84]. The reported closed and open conformations of the LRRC8A channel indicated the coupled dilation of cytoplasmic LRRs and the channel pore, suggesting a mechanism for channel gating by an internal stimulatory signal.

Notably, the transmembrane domain is structurally related to connexin proteins that form gap junctions between cells for direct intercellular transport of 2′3′-cGAMP, implying that some essential structural features might be shared between connexins and LRRC proteins for transport of 2′3′-cGAMP. Despite these similarities, notable differences also exist, given that gap junction channels form wide pores exhibiting poor selectivity, thereby allowing diffusion of larger substrates, while VRACs are selective for smaller anions, while showing negligible conductance for cations.

Recently, the above structural studies on homomeric LRRC8A hexameric channels [84] have been extended to their heteromeric LRRC8A/C hexameric counterparts [104]. The heteromeric channel is composed of four A subunits that cluster in the conformation preferred in the homomeric topology, while two C subunits show greater flexibility. It has been proposed that the flexible C subunits may perturb the tight interaction of the four subunit A cluster that is reminiscent of the closed state, thereby facilitating the activation of the channel. Notably, mutational studies highlight the role of charged residues positioned at the narrowest constriction of the homomeric VRAC channel as a pore determinant of heteromeric VRAC counterpart [106].

Given that the structure of hexameric LRRC8 is only available in the apo state (Figure 5A), insights into 2’3’-cGAMP recognition and transport must await the structure determination of the 2’3’-cGAMP-LLRC8 complex, towards a more precise understanding of the relationships between homomeric and heteromeric LRRC8 protein scaffolds in the transport of CDN second messengers. It is noteworthy that although respective CDN-bound structures are lacking, researchers predicted that LLRC8A may plays a crucial role in 2′3′-cGAMP recognition by noting that the LRRC8A channel is gated by an arginine residue (R103) that is not observed in LLRC8C/E, and by also considering the negatively charged phosphodiester ring of 2′3′-cGAMP. This prediction was confirmed by evaluating the extracellular cGAMP response upon rescued expression of wildtype proteins and R103L substituted LRRC8A in the corresponding knockout telomerase-immortalized human microvascular endothelial (TIME) cells, supporting the idea that LLRC8A R103 regulates channel permeation through charge-charge interactions [61].

Further studies are also needed to investigate the potential of LRRC8A to mediate direct bidirectional transport dependent on 2’3’-cGAMP concentration gradient.

P2X7R importer

The ATP-gated P2X purinergic plasma membrane receptor family proteins are trimeric, non-selective cation channels highly expressed in immune cells and which are activated by extracellular ATP for cell communication [85]. P2X family member P2X7R importer has been shown to play a crucial role in the immune response to infection and inflammation amongst most cells of innate and adaptive immunity [86]. P2X7R requires a high concentration of extracellular ATP that is often present in the inflammatory microenvironment for activation. The increased extracellular ATP concentrations significantly expand the ATP-gated P2X7R channel, which allows nanometer-sized molecules to pass directly through the cell membrane. A recent study reported that the addition of ATP and 2’3’-cGAMP in the cell medium increased the phosphorylation of TBK1 and IRF3 in cultured bone marrow-derived macrophages (BMDMs) in a P2X7R-dependent manner [60]. The study also confirmed the role of P2X7R in ATP-enhanced 2’3’-cGAMP importation by using BMDMs from P2X7R knockout mice. P2X7R allows 2’3’-cGAMP to enter host immune cells, which enhances innate immune sensing of tumor cells and production of type I IFNs. While the direct biochemical evidence for P2X7R-driven CDN transport remains to be established, there are other possibilities such as the potential link between P2X7R activity and gap junctions or SLC19A1 activity, in relation to CDN transport. The study suggests 2’3’-cGAMP as an extracellular danger signal that coordinates with extracellular ATP to activate STING-dependent type I IFN responses.

Structural studies undertaken on the P2X7R importer outlined in Box 5 and Figure 5C identify a trimeric topology adopted by P2X7R to generate a channel that is closed in the apo state and open in the ATP-bound state [87]. The structure of P2X7 receptor contains a pair of cytoplasmic elements named the C-cys anchor and the cytoplasmic ballast, that play a role in receptor desensitization. The involvement of the P2X7 receptor subtype in apoptosis, inflammation, and tumor suppression pathways, makes it an attractive pharmacological target for anti-inflammatory and anti-cancer therapies [86].

Box 5. Structural biology of P2X7 importer.

The reported cryo-EM structures of rat P2X7 receptor in apo (closed) and ATP-bound (open) states [87] provide basic information about the overall fold of the receptor, subunit–subunit interactions, as well as hints as to the location of the ATP binding site (Figure 5C). The overall assembly and subunit folding of the rat P2X7 receptor shares a chalice-like trimeric architecture, consistent with those of other previously determined P2X structures solved using x-ray crystallography [107–109]. The monomer of P2X7 comprises a large hydrophilic extracellular domain, two transmembrane helices, and a cytoplasmic ballast, representing the shape of a leaping dolphin. The extracellular domain in the trimeric state makes extensive subunit–subunit contacts in the upper regions. Fewer contacts are observed in the regions proximal to the TM domains, forming cavities between subunits for binding of the agonist ATP. The zinc ion-containing cytoplasmic ballast has a GDP binding site in the open state, yet the role of GDP requires further experimentation. Another cytoplasmic element, the C-cys anchor, uses palmitoyl groups to anchor the pore-lining helix to the membrane, thereby preventing desensitization [87].

A future challenge will be to extend the structural studies to the P2X7R channel in the 2′3′-cGAMP bound state to localize both its CDN binding site and potential conformational changes associated with complex formation. Such an analysis will be crucial for a precise understanding of the mechanism of ATP-enhanced 2′3′-cGAMP uptake and positioning mediated by P2X7R, as well as aspects of ligand permeability and cell-type specificity, leading to the development of new intervention strategies against inflammatory diseases.

EXPORTERS OF EXTRACELLULAR 2′3′-cGAMP

The above studies emphasized discoveries related to importers of 2′3′-cGAMP. A fundamental question addresses whether there are specific exporters for 2′3′-cGAMP to exit cells that produce it. We report below on ATP-binding exporter multidrug resistance protein 1 (MRP1).

MRP1 exporter

Recently, MRP1, also known as ATP-binding cassette transporter 1 (ABCC1), was identified to be a mediator of direct, ATP-dependent 2′3′-cGAMP export [88]. MRP1 was first identified from drug-resistant lung cancer cells that did not express the P-glycoprotein (P-gp) transporter [89]. It was later demonstrated to mediate the export of a large range of endogenous and exogenous macromolecules, including cysteinyl leukotriene C4 (LTC4), the conjugated estrogen E217bG, glutathione (GSH), anthracycline chemotherapeutic drugs, as well as antibiotics [90]. Inspired by the prior identification of energy-dependent transporters that mediate the transport of CDNs in bacteria [91], the efforts next focused on looking for the potential exporter of 2′3′-cGAMP among the ATP-binding cassette family of transmembrane proteins. Systematic screening of inhibitors for distinct classes of ABC transporters by measuring their effect on 2′3′-cGAMP export in bone-marrow-derived macrophages (BMMs), resulted in the discovery that compound MK-571, the inhibitor of MRP1, contributed to the blockade of 2′3′-cGAMP export [88,90]. The authors further revealed that MRP1 mediates direct, ATP-dependent, GSH-independent export of 2′3′-cGAMP to limit cell-intrinsic STING responses and restrain cGAS-dependent autoimmunity in human cells and mouse models.

Structural studies undertaken on MRP1 exporter outlined in Box 6 and Figure 6 have defined monomeric alignments in the apo-, LTC4 substrate-bound, and ATP-bound states, as well as structural principles associated with substrate LTC4 recognition [92,93]. The kinetic cycle of the MRP1 ABC transporter has been characterized further by single-molecule fluorescence spectroscopy studies [94]. Notably, bound substrate stimulates ATP hydrolysis by accelerating the inward-facing to outward-facing conformational transition. In addition, dissociation of the dimeric alignment of the NBD domains constitutes the rate-limiting step of the transport cycle.

Box 6. Structural biology of MRP1 exporter.

To date, cryo-EM structures have been reported for bovine MRP1 (Figure 6A) in the apo- (Figure 6B, left panel; inward-facing conformation), LTC4-substate-bound (Figure 6B, middle panel; inward-facing conformation), and ATP-bound (Figure 6B, right panel; outward-facing conformation) states [92,93]. The molecular structure of MRP1 has an additional N-terminal transmembrane domain helical bundle (TMD0) that contains five transmembrane helices followed by an interfacial portion known as the lasso motif. The majority of the MRP1 fold forms a canonical transporter core similar to other inward-facing ABC exporters, such as P-gp [110]. The 12 transmembrane helices from TMD1 and TMD2 are arranged into two pseudo-symmetric bundles. A large transmembrane vestibule at the interface of the two helical bundles opens to the cytoplasm and penetrates halfway into the lipid bilayer. The two nucleotide binding domains (NBDs) are extensively separated on proceeding down into the cytoplasm.

The cryo-EM reconstruction of the LTC4-bound form of MRP1 defines the position of LTC4 and provides insights into the molecular mechanism of substrate recognition. LTC4 binds MRP1 inside the transmembrane cavity between the two TM bundles in the membrane (Figure 6B, middle panel). MRP1 in an inward facing resting conformation forms a single bipartite binding pocket that can accommodate substrates containing a spectrum of chemical topologies. Substrate binding induces local and global conformational changes in the two halves of the transporter, with closer movement priming MRP1 for ATP hydrolysis. The structure of MRP1 in the ATP-bound state reveals that ATP binding opens the transport pathway to the extracellular space (Figure 6B, right panel) and reconfigures the substrate-binding site such that it relinquishes its affinity for the substrate, indicating that the substrate can be released before ATP hydrolysis.

A future challenge will be to extend the structural studies to MRP1 channels in the 2′3′-cGAMP bound state to check whether the 2’3’-cGAMP occupies the substrate binding pocket and whether 2’3’-cGAMP and ATP can be bound at the same time or are mutually exclusive. Such studies can help to further understand the mechanism of MRP1-mediated 2′3′-cGAMP export and provide insights into potential strategies for modulating this process, with possible implications for the treatment of autoimmune diseases and cancer.

CONCLUDING REMARKS

Future Challenges

This review summarizes and contrasts recent advances in 2′3′-cGAMP transport across membranes from a structure-function perspective. Guided by currently available structural information, we discuss structure-based mechanistic insights into 2′3′-cGAMP mediated intercellular immune signal transmission. It is noteworthy that 2′3′-cGAMP is involved in a complex regulatory network including a variety of carriers and channels. As such, a detailed understanding of the mechanisms underlying 2′3′-cGAMP transport is essential for developing effective therapeutic strategies that can precisely modulate this network, with implications for the treatment of inflammatory, autoimmune, and degenerative diseases. Moreover, the substantial barriers to drug development targeting the cGAS-STING pathway include multiple levels of complexity associated with regulation of cGAS-STING signaling, such as the role of specific transporters and hydrolases, that can modulate the cGAS-STING pathway associated with second messenger CDN transport and/or degradation. These different pathways are part of a complex regulatory network associated with the immune response, with ongoing challenges requiring an improved understanding from a structural and functional perspective of regulatory factors that mediate the cGAS-STING pathway.

The candidate drugs targeting the delivery of 2′3′-cGAMP [41], as well as inhibitors of 2′3′-cGAMP degradation [41,54,95], represent a promising strategy that will require an understanding of their mode pf action at the mechanistic level, thereby offering new therapeutic opportunities towards the treatment of cancer and autoimmune diseases. As an example, the available detailed structural information on antifolate PMX targeted to SLC19A1 and SLC46A1 offers additional opportunities for further structure-guided optimization of inhibitors that impact CDN transport by targeting the CDN binding channel of these transporters.

In the past few years, extensive interest has focused on efforts aimed at using synthetic CDN derivatives and non-nucleotide small molecules towards therapeutically targeting the cGAS-STING pathway [41]. Thus, inhibitors of cGAS have been identified that either target the catalytic pocket for 2′3′-cGAMP generation or alternately disrupt DNA binding necessary for cGAS activation. In addition, both STING agonists and antagonists have been identified that either non-covalently target the CDN-binding site within STING or alternately serve as antagonists by covalently targeting the Cys91 palmitoylation site near the transmembrane domain of STING. A recent structure-guided study showed that C53, a small molecule acting as an agonist of STING, targets a novel pocket in the membrane-spanning domain of STING [96]. This finding suggests a new activation mechanism for STING, setting the stage for structure-guided optimization of C53, based on the intermolecular interactions and conformational changes on complex formation [97]. Currently, several candidate small molecules are undergoing clinical trials as either vaccine adjuvants or for immunotherapies targeting cancers; the focus lies on retaning minimum levels of inhibition of the cGAS-STING pathway required for therapeutic benefit, without significantly impacting on other essential compensatory innate immune regulatory pathways.

Regarding transport mechanisms, the best studied CDN transport system to date has been the anionic exchanger SLC19A1 for which structural information is available for transport of CDNs, as well as folates/antifolates, bound in distinct pockets within the same substrate channel in an inward-open conformation (Figure 4B, C) [78,80]. Nevertheless, much remains to be structurally characterized about distinct conformational states (inward-open, outward-open and occluded) associated with the transport cycle, as well as the location and composition of the binding pockets for CDNs and folates/antifolates in these alternate states. Such an analysis would provide insights into the driving force and energetics dictating unidirectionality of 2’3’-cGAMP transport. This should be feasible using different nanodisc-reconstituted SLC19A1-Fab systems, given that a structure is now available for PMX bound to SLC46A1 in the outward-open conformation [79]. In addition, in the case of SLC19A1, additional research is required for the development of a molecular level understanding (binding pockets and states) on the counter-transport of either organic anions (nucleotides and thiamine pyrophosphates) or their inorganic counterparts (phosphate and chloride), in an effort to understand the energetics that couple the import of CDNs and folates/antifolates with anion export across the cell membrane. Further, cyclic trinucleotides have been identified as second messengers in bacteria [98–100], and it is not clear at this time whether they are involved in transport by SLC19A1 across the cell membrane, and if so, whether they do so as monomers or dimers.

There are presently no structures of 2’3’-cGAMP bound to importers LRRC8 and P2X7R, or exporter MRP1; this information will be necessary to locate binding pocket(s), and veer towards an understanding of the molecular principles underlying the specificity and cell-type of molecular recognition, and the nature of the anticipated conformational changes associated with complex formation and transport.

Finally, it remains unclear whether pathogens can evolve proteins capable of blocking import/export of 2’3’-cGAMP from producer cells to surrounding bystander cells, thereby bypassing pathogen factors targeting STING in the infected cell. Also, a future challenge relates to the impact of pathogens on the efficiency, cell-type and species specificity of CDN transport (see outstanding questions).

OUTSTANDING QUESTIONS.

To what extent can structural studies validate functional support for 2’3’-cGAMP transport by LRRC8 and P2X7R importers and by MRP1 exporter? These efforts should be geared towards elucidating the principles underlying recognition specificity, and the nature of the anticipated conformational changes associated with complex formation and transport.

Can structural studies identify a range of states (inward-open, outward-open and occluded) of the 2’3’-GAMP channels of interest? What triggers and regulates the switch between distinct states of the channel?

What is the driving force dictating the unidirectional flow of 2’3’-cGAMP through the channel? Can the efficiency of transport be modulated by selective mutations and/or chemical modifications impacting on pore dimensions of the channel?

What Is the energetic landscape that couples the import of CDNs and folates/antifolates with the export of organic anions (nucleotides and thiamine pyrophosphates) and inorganic counterparts (phosphate and chloride) across the cell membrane of the SLC19A1 channel?

What is the physical basis for lipid-based stabilization of gap junctions and to what extent is it dependent on the type and stoichiometry of lipids? Is there an impact by delipidation enzymes on the transport process?

Given that cyclic dinucleotide second messengers form intertwined compact dimers when positioned within the inward-open topology of the SLC19A1 channel, do larger cyclic trinucleotide bacterial second messengers adopt monomeric or dimeric folding topologies if transported through the channel?

Can available structures of folates and anti-folates bound within the central segment of the SLC19A1 channel provide insights into the development of next-generation small molecule agonists and antagonists impacting transport?

Highlights.

Systematic structural studies of gap junctions, host defense peptides, importers, and exporters, have highlighted insights into channel stoichiometries and topologies, pore diameters, and functional states mediating second messenger 2’3’-cGAMP and CDN transport.

A comprehensive structural investigation of a range of 2’3’-cGAMP transporters have outlined the diversity of intracellular, membrane, and extracellular components constituting channel architecture, thereby defining channels with distinct functional capabilities.

The structures of CDNs including 2’3’-cGAMP bound to the SLC19A1 channel have highlighted the intertwined compact dimeric second messenger fold and its positioning in an inward-open channel, thereby opening opportunities for investigation of the corresponding outward-open and occluded states, towards a molecular understanding of the transport cycle.

The positioning of monomeric folates and anti-folates in the interior of the SLC19A1 channel have provided a foundation for future design and optimization of agonists and antagonists impacting on 2’3’-cGAMP transport, with therapeutic implications for inflammatory and immune-associated pathologies.

SIGNIFICANCE.

There are recent insights into the regulated channel-mediated transport of 2′3′-cGAMP associated with the cGAS-STING signaling pathway in inflammation, from a structural perspective and a functional context. This has defined an underlying mechanism for 2′3′-cGAMP as an ‘immunotransmitter’ that mediates intercellular communication. Structures are available for apo-, 2’3’-cGAMP-, folate- and antifolate-bound states of the SLC19A1 channel, providing insights into both the transport cycle and approaches for generation and testing of small molecule agonists and antagonists impacting on second messenger transport.

Acknowledgments

DJP was supported by funds from the NIH (GM129430 and AI451507), the Maloris Foundation and Memorial Sloan-Kettering Cancer Center Core grant P30 CA008748. WX was supported by funds from the National Natural Science Foundation of China Grant 82151216.

Glossary

cGAS-STING innate immune pathway: Consists of three major components - the ‘sensor’ cyclic GMP-AMP synthase (cGAS), the ‘second messenger’ cyclic 2’3’-GMP-AMP (2′3′-cGAMP), and the ‘adaptor’ stimulator of interferon genes (STING)
Liquid-like condensates: type of biomolecular condensate with liquid-like properties, allowing them to rapidly exchange molecules with the surrounding environment. Liquid-like condensates can concentrate proteins and other biomolecules in specific regions of cells to play important biological roles
Aicardi-Goutières syndrome: rare genetic disorder affecting the brain, skin, and immune system; caused by mutations in genes involved in the regulation of the immune system, including the cGAS/STING signaling pathway. There is currently no cure, but treatment focuses on managing symptoms and preventing complications
Cyclic dinucleotides (CDNs): Secondary signaling molecules in bacteria and mammalian cells. Unlike bacterial CDNs (such as 3’3’-cGG, 3’3’-cAA and 3’3’-cGAMP) which are composed of two 3′-5 linkages, the mammalian CD-NTase cGAS produces 2′3′-cGAMP, which contains mixed 2′-5′ (GpA step) and 3′-5′ (ApG step) phosphodiester linkages
Gap junctions: transmembrane family of connexin proteins form intercellular channels, termed gap junctions, for cell-to-cell communication. Such gap junctions span the cytoplasms of neighboring cells to facilitate the passage of ion-mediated electrical signals, as well as small molecules ranging from second messengers to therapeutic agents. The intercellular channel functions of such gap junctions are dependent on a mosaic of lipid-protein interactions
Human host defense peptide LL-37: The human cathelicidin antimicrobial peptide gene encodes the precursor protein CAP (1–170 aa). Cleavage of CAP results in the production of the human host defense peptide LL-37 (corresponding to amino acids 134–170 of CAP), which is further processed during its secretion
SLC19A1 and SLC46A2: Both SLCs transporters are known as importers involved in the transport of folate nutrients and antifolate therapeutics. Recently, they were also shown to be importers of 2’3’-cGAMP
Volume-regulated anion channels (VRACs): exhibit variable selectivity and conductance of chloride and other anions, including exchange of diverse osmolytes, to maintain a constant cell volume in response to osmotic stress
Leucine-rich repeat-containing protein 8A (LRRC8A): acts as the ubiquitously essential component of VRAC across cell types. LRRC8A and its paralogs (types B, C, D and E) form heteromeric channels to conduct the complex function of VRACs in a cell- and species-specific manner
P2X7R importer: ATP-gated P2X7R purinergic plasma membrane receptor which forms non-selective cation channels that play a crucial role in the immune response to infection and inflammation amongst most cells of innate and adaptive immunity
Multidrug resistance protein 1 (MRP1): functions as a transporter that mediates direct, ATP-dependent 2′3′-cGAMP export of a large range of endogenous and exogenous macromolecules
Nanodisc-reconstituted SLC19A1-Fab system: Involves reconstituting SLC19A1 into a nanodisc, which is a small disc-shaped lipid bilayer that mimics the natural membrane environment of the transporter. The system also contains a Fab antibody fragment that binds specifically to SLC19A1
Pterin chromophores: class of organic molecules that contain a pterin aromatic ring system; heterocyclic compounds found in many biological systems, including complexes composed of proteins, DNA, and RNA

Footnotes

Declaration of Interests

WX and DJP declare no competing interests.

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PERMALINK

Structure-based mechanisms of 2′3′-cGAMP intercellular transport in the cGAS-STING immune pathway

Wei Xie

Dinshaw J Patel