Abstract
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.
Highlights
Nucleosides play crucial roles in cell homeostasis, functioning as nucleotide precursors [1] and signaling molecules [2]
We purified full-length wild-type hCNT3 and CNT3ins with monodispersed peaks in detergent micelles (S1 Fig). Both proteins were eluted from size-exclusion chromatography at approximately 14 mL, which suggested that hCNT3 and CNT3ins present identical oligomerization states
Considering the trimeric architecture of bacterial concentrative nucleoside transporter (CNT) [18] and the cross-linking assay of hCNT3 [28], we speculated that purified hCNT3 and CNT3ins existed as trimers in detergent micelles
Summary
Nucleosides play crucial roles in cell homeostasis, functioning as nucleotide precursors [1] and signaling molecules [2]. Nucleosides can be synthesized de novo or via a salvage pathway. Many human cells are unable to synthesize nucleosides via the de novo biosynthetic pathway [3]. To achieve nucleoside homeostasis, the nucleoside transporter facilitated nucleoside salvage pathway is of great importance [4]. Solute carrier (SLC) transporters facilitate the transport of multiple substrates and play important roles in physiological processes [7]. Structural investigations are vital for understanding the transport mechanism of SLC transporters [8,9,10]. Several nucleoside transporters in mammals have been identified and classified into two SLC transporter families: concentrative nucleoside transporters (CNTs, the SLC28 family) and equilibrative nucleoside
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