Recognition of cyclic dinucleotides and folates by human SLC19A1

Cyclic dinucleotides (CDNs) are ubiquitous signalling molecules in all domains of life^1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, through cyclic GMP-AMP synthase after detecting cytosolic DNA signals^3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogues, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses^8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is dependent on the solute carrier SLC19A1^22,23. Moreover, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics^24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs, folate and antifolate is unclear. Here we report cryo-electron microscopy structures of human SLC19A1 (hSLC19A1) in a substrate-free state and in complexes with multiple CDNs from different sources, a predominant natural folate and a new-generation antifolate drug. The structural and mutagenesis results demonstrate that hSLC19A1 uses unique yet divergent mechanisms to recognize CDN- and folate-type substrates. Two CDN molecules bind within the hSLC19A1 cavity as a compact dual-molecule unit, whereas folate and antifolate bind as a monomer and occupy a distinct pocket of the cavity. Moreover, the structures enable accurate mapping and potential mechanistic interpretation of hSLC19A1 with loss-of-activity and disease-related mutations. Our research provides a framework for understanding the mechanism of SLC19-family transporters and is a foundation for the development of potential therapeutics.