Recognition of cyclic dinucleotides and folates by human SLC19A1

Cyclic dinucleotides (CDNs) are ubiquitous signaling molecules in all domains of life1,2. Mammalian cells produce one CDN, 2'3'-cGAMP, by cyclic GMP-AMP synthase upon detecting cytosolic DNA signals3-7. 2'3'-cGAMP, as well as bacterial and synthetic CDN analogs, can act as second messengers to activate stimulator of interferon genes (STING) and elicit broad downstream responses8-21. Extracellular CDNs must traverse the cell membrane to activate STING, a process that is critically dependent on the solute carrier SLC19A122,23. In addition, SLC19A1 represents the major transporter for folate nutrients and antifolate therapeutics24,25, thereby placing SLC19A1 as a key factor in multiple physiological and pathological processes. How SLC19A1 recognizes and transports CDNs and folate/antifolate is unknown. 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. Structural and mutagenesis results demonstrate that hSLC19A1 utilizes 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, while folate or antifolate binds as a monomer and occupies a distinct pocket of the cavity. Moreover, the structures allow accurate mapping and potential mechanistic interpretation of loss-of-activity and disease-related mutations of hSLC19A1. Our work provides a framework for understanding the mechanism of SLC19 family transporters and serves as a foundation for the development of potential therapeutics.