Sodium Coupling and Nucleoside Specificity of a Concentrative Nucleoside Transporter

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Concentrative nucleoside transporters (CNTs) are ion-coupled membrane transporters that utilize ion gradients to transport nucleosides and nucleoside-derived anticancer and antiviral drugs into cells. Humans have three subtypes of CNTs with varying nucleoside and nucleoside-drug specificity. The crystal structure of a CNT from Vibrio cholerae (vcCNT) in complex with uridine revealed the overall architecture, the nucleoside-binding site, and the putative sodium-binding site of this class of transporter 1.In our follow-up studies, we have addressed two issues: the role of sodium in nucleoside transport and the substrate specificity of vcCNT. With regard to the role of sodium in nucleoside transport, the hypothesis that we gained from the structure is that sodium binding stabilizes the nucleoside-binding site thereby increasing its affinity for nucleosides. To test this structural hypothesis, we performed mutagenesis, isothermal titration calorimetry, and radioactive nucleoside uptake experiments. Our results are consistent with our structural hypothesis. To dissect the substrate specificity of vcCNT, we first observed that the nucleoside-binding site of vcCNT is very similar to hCNT3 (>90% sequence identity). As a result, our functional studies showed that vcCNT displays similar substrate specificity as hCNT3. To understand the structural basis of nucleoside and nucleoside-drug specificity, we have performed crystallographic and functional studies of vcCNT in complex with several different nucleosides and nucleoside-derived drugs. Our results provide a structural basis for the substrate specificity of vcCNT and hCNT3.Reference1. Zachary Johnson, Cheom-Gil Cheong, and Seok-Yong Lee, Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 A. Nature. 2012, Mar 11;483(7390):489-93.