Structure and mechanism of human sphingosine-1-phosphate transporter MFSD2B

Sphingosine-1-phosphate (S1P) is an essential signaling lipid that maintains vascular integrity and regulates immune cell trafficking. The major facilitator superfamily domain–containing protein 2B (MFSD2B) serves as the main S1P exporter in red blood cells and platelets; however, its structure and transport mechanism are unclear. Here, we report the 3.0 Å cryo-EM structure of human MFSD2B bound to S1P. S1P is captured in a distinctive binding state, deeply buried within the C-domain, with its sphingoid tail accommodated by a hydrophobic pocket and its phosphate group coordinated by a cluster of polar residues within the transporter’s cavity. Mutagenesis and molecular dynamics simulations identify the TM2/TM11 lateral opening as the primary pathway for S1P translocation, with key charged residues acting as sequential anchors during transport. Furthermore, we demonstrate that MFSD2B functions as a uniporter, and that subtle rewiring of local charge networks can alter its coupling mechanism. Our work provides a molecular framework for understanding S1P transport mediated by MFSD2B in hematopoietic cells.