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Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters.

<div class='caption-title'>(A) The vcCNT-7C8C trimer viewed from within the plane of the membrane. The location of the membrane is marked by rectangles. The scaffold domain of one protomer is colored light blue, and the transport domain is colored red, blue, orange, cyan, wheat, and brown. The other two protomers are colored gray. Uridine is shown bound to each protomer in stick representation. The nucleoside-binding site is delineated with dashed lines. vcCNT-7C8C functions similarly to wild type (Figure 1—figure supplement 1). (B) The vcCNT-7C8C protomer. The structure is rotated 120° about the trimer axis relative to A, zoomed in, and the other two protomers have been removed for clarity. (C) Nucleoside-binding site. Amino acid residues that interact with the uridine are labeled and shown in stick representation and were used for sequence identity calculation with hCNTs. Hydrogen bonds are shown as dashed lines. The uracil base is marked with a blue box, and the ribose is marked with a gray box. For a stereo view of the electron density in the nucleoside-binding site, see Figure 1—figure supplement 2. (D) Fluorescence titration of vcCNT with uridine. Uridine was titrated into solution containing vcCNT and the fluorescent nucleoside pyrrolo-cytidine, anisotropy was measured, and data were fit to a single-site competitive binding model to obtain a KD of 36 ± 3 μM (mean ± SEM, n = 3 measurements).</div>
<div class='caption-title'>Wild-type (WT) and 7C8C vcCNT were reconstituted into lipid vesicles and assessed for their ability to support the uptake of 2 μM of 3H-labeled nucleosides as described (Johnson et al., 2012). The data shown represent 2 min timepoints. Empty vesicles were included as a negative control.</div>
<div class='caption-title'>The nucleoside-binding site of vcCNT-7C8C bound to uridine is shown in stereo in stick representation in the same orientation and colored as in Figure 1C. Uridine is yellow and the red spheres are water molecules. The resolution is 2.1 Å and density shown is from a 2Fo–Fc electron density map contoured at 1σ.</div>
Johnson ZL, Lee JH, Lee K, Lee M, Kwon DY, Hong J, Lee SY
OpenAccess logo Elife 3 e03604 (2014)
Related entries: 4pb1, 4pd5, 4pd6, 4pd7, 4pd8, 4pd9, 4pda

Cited: 31 times
EuropePMC logo PMID: 25082345

Abstract

Concentrative nucleoside transporters (CNTs) are responsible for cellular entry of nucleosides, which serve as precursors to nucleic acids and act as signaling molecules. CNTs also play a crucial role in the uptake of nucleoside-derived drugs, including anticancer and antiviral agents. Understanding how CNTs recognize and import their substrates could not only lead to a better understanding of nucleoside-related biological processes but also the design of nucleoside-derived drugs that can better reach their targets. Here, we present a combination of X-ray crystallographic and equilibrium-binding studies probing the molecular origins of nucleoside and nucleoside drug selectivity of a CNT from Vibrio cholerae. We then used this information in chemically modifying an anticancer drug so that it is better transported by and selective for a single human CNT subtype. This work provides proof of principle for utilizing transporter structural and functional information for the design of compounds that enter cells more efficiently and selectively.

Reviews - 4pb2 mentioned but not cited (2)

  1. Elevator-type mechanisms of membrane transport. Garaeva AA, Slotboom DJ. Biochem Soc Trans 48 1227-1241 (2020)
  2. Toward a Molecular Basis of Cellular Nucleoside Transport in Humans. Wright NJ, Lee SY. Chem Rev 121 5336-5358 (2021)

Articles - 4pb2 mentioned but not cited (2)

  1. Structural basis of nucleoside and nucleoside drug selectivity by concentrative nucleoside transporters. Johnson ZL, Lee JH, Lee K, Lee M, Kwon DY, Hong J, Lee SY. Elife 3 e03604 (2014)
  2. The Structure and Mechanism of Drug Transporters. Roberts AG. Methods Mol Biol 2342 193-234 (2021)


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  1. SLC transporters as therapeutic targets: emerging opportunities. Lin L, Yee SW, Kim RB, Giacomini KM. Nat Rev Drug Discov 14 543-560 (2015)
  2. Multifaceted roles of extracellular DNA in bacterial physiology. Vorkapic D, Pressler K, Schild S. Curr Genet 62 71-79 (2016)
  3. Polypharmacology of conformationally locked methanocarba nucleosides. Jacobson KA, Tosh DK, Toti KS, Ciancetta A. Drug Discov Today 22 1782-1791 (2017)
  4. Recent advances on the inhibition of human solute carriers: Therapeutic implications and mechanistic insights. Wright NJ, Lee SY. Curr Opin Struct Biol 74 102378 (2022)
  5. An Analysis of Mechanisms for Cellular Uptake of miRNAs to Enhance Drug Delivery and Efficacy in Cancer Chemoresistance. Grixti JM, Ayers D, Day PJR. Noncoding RNA 7 27 (2021)
  6. Disruption of small molecule transporter systems by Transporter-Interfering Chemicals (TICs). Nicklisch SCT, Hamdoun A. FEBS Lett 594 4158-4185 (2020)
  7. Novel molecular mechanisms of doxorubicin cardiotoxicity: latest leading-edge advances and clinical implications. Robert Li Y, Traore K, Zhu H. Mol Cell Biochem (2023)

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  1. The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism. Mulligan C, Fenollar-Ferrer C, Fitzgerald GA, Vergara-Jaque A, Kaufmann D, Li Y, Forrest LR, Mindell JA. Nat Struct Mol Biol 23 256-263 (2016)
  2. Structures of human ENT1 in complex with adenosine reuptake inhibitors. Wright NJ, Lee SY. Nat Struct Mol Biol 26 599-606 (2019)
  3. Repeat-swap homology modeling of secondary active transporters: updated protocol and prediction of elevator-type mechanisms. Vergara-Jaque A, Fenollar-Ferrer C, Kaufmann D, Forrest LR. Front Pharmacol 6 183 (2015)
  4. Visualizing conformation transitions of the Lipid II flippase MurJ. Kuk ACY, Hao A, Guan Z, Lee SY. Nat Commun 10 1736 (2019)
  5. Visualizing multistep elevator-like transitions of a nucleoside transporter. Hirschi M, Johnson ZL, Lee SY. Nature 545 66-70 (2017)
  6. Functional characterization of human equilibrative nucleoside transporter 1. Huang W, Zeng X, Shi Y, Liu M. Protein Cell 8 284-295 (2017)
  7. Nucleoside uptake in Vibrio cholerae and its role in the transition fitness from host to environment. Gumpenberger T, Vorkapic D, Zingl FG, Pressler K, Lackner S, Seper A, Reidl J, Schild S. Mol Microbiol 99 470-483 (2016)
  8. A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis. Ramírez-Guadiana FH, Meeske AJ, Rodrigues CDA, Barajas-Ornelas RDC, Kruse AC, Rudner DZ. PLoS Genet 13 e1007015 (2017)
  9. Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS. Kim JW, Kim S, Kim S, Lee H, Lee JO, Jin MS. Sci Rep 7 2548 (2017)
  10. Cryo-EM structure of the human concentrative nucleoside transporter CNT3. Zhou Y, Liao L, Wang C, Li J, Chi P, Xiao Q, Liu Q, Guo L, Sun L, Deng D. PLoS Biol 18 e3000790 (2020)
  11. Rational Design of Nucleoside-Bile Acid Conjugates Incorporating a Triazole Moiety for Anticancer Evaluation and SAR Exploration. Navacchia ML, Marchesi E, Mari L, Chinaglia N, Gallerani E, Gavioli R, Capobianco ML, Perrone D. Molecules 22 E1710 (2017)
  12. Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine. Forciniti S, Dalla Pozza E, Greco MR, Amaral Carvalho TM, Rolando B, Ambrosini G, Carmona-Carmona CA, Pacchiana R, Di Molfetta D, Donadelli M, Arpicco S, Palmieri M, Reshkin SJ, Dando I, Cardone RA. Int J Mol Sci 22 E29 (2020)
  13. Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance. Mulinta R, Yao SYM, Ng AML, Cass CE, Young JD. J Biol Chem 292 9505-9522 (2017)
  14. The ups and downs of elevator-type di-/tricarboxylate membrane transporters. Sauer DB, Wang B, Sudar JC, Song J, Marden J, Rice WJ, Wang DN. FEBS J 289 1515-1523 (2022)
  15. Homology Modeling of Human Concentrative Nucleoside Transporters (hCNTs) and Validation by Virtual Screening and Experimental Testing to Identify Novel hCNT1 Inhibitors. Kumar Deokar H, Barch HP, Buolamwini JK. Drug Des 6 146 (2017)
  16. Rosetta Broker for membrane protein structure prediction: concentrative nucleoside transporter 3 and corticotropin-releasing factor receptor 1 test cases. Latek D. BMC Struct Biol 17 8 (2017)
  17. Survey of ribose ring pucker of signaling nucleosides and nucleotides. Salmaso V, Jacobson KA. Nucleosides Nucleotides Nucleic Acids 39 322-341 (2020)
  18. Concentrative Nucleoside Transporter 3 Is Located on Microvilli of Vaginal Epithelial Cells. Webster P, Saito K, Cortez J, Ramirez C, Baum MM. ACS Omega 5 20882-20889 (2020)
  19. Molecular basis for substrate recognition by the bacterial nucleoside transporter NupG. Wang C, Xiao Q, Duan H, Li J, Zhang J, Wang Q, Guo L, Hu J, Sun B, Deng D. J Biol Chem 296 100479 (2021)
  20. Comparison of the diastereoisomeric excess of uridine, inosine and adenosine cyanohydrins determined by HPLC-DAD and 1H NMR. Martínez-Casares RM, Pérez Méndez HI, Manjarrez Alvarez N, Solís Oba A, Hernández Vázquez L, López-Luna A. Nucleosides Nucleotides Nucleic Acids 36 652-665 (2017)


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