Qixiang Zhang,et al.

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.