Concentrative nucleoside transporters (CNTs), members of the solute carrier (SLC) 28 transporter family, facilitate the salvage of nucleosides and therapeutic nucleoside derivatives across the plasma membrane. Despite decades of investigation, the structures of human CNTs remain unknown. We determined the cryogenic electron microscopy (cryo-EM) structure of human CNT (hCNT) 3 at an overall resolution of 3.6 Å. As with its bacterial homologs, hCNT3 presents a trimeric architecture with additional N-terminal transmembrane helices to stabilize the conserved central domains. The conserved binding sites for the substrate and sodium ions unravel the selective nucleoside transport and distinct coupling mechanism. Structural comparison of hCNT3 with bacterial homologs indicates that hCNT3 is stabilized in an inward-facing conformation. This study provides the molecular determinants for the transport mechanism of hCNTs and potentially facilitates the design of nucleoside drugs.

浓缩核苷转运蛋白（CNTs）是溶质载体（SLC）28转运蛋白家族的成员，促进了整个细胞膜中核苷和治疗性核苷衍生物的抢救。尽管进行了数十年的研究，但人类CNT的结构仍然未知。我们确定了人类CNT（hCNT）3的低温电子显微镜（cryo-EM）结构，整体分辨率为3.6。与其细菌同源物一样，hCNT3呈现三聚体结构，带有额外的N端跨膜螺旋，以稳定保守的中央结构域。底物和钠离子的保守结合位点揭示了选择性核苷转运和独特的偶联机理。hCNT3与细菌同源物的结构比较表明，hCNT3以向内构象稳定。