Cysteine cross-linking defines the extracellular gate for the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1)

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Abstract

Equilibrative nucleoside transporters are a unique family of proteins that enable uptake of nucleosides/nucleobases into a wide range of eukaryotes and internalize a myriad of drugs used in the treatment of cancer, heart disease, AIDs, and parasitic infections. In previous work we generated a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani, using ab initio computation. The model suggested that aromatic residues present in transmembrane helices 1, 2, and 7 interact to form an extracellular gate that closes the permeation pathway in the inward-open conformation. Mutation of residues Phe-48TM1 and Trp-75 TM2 abrogated transport activity, consistent with such prediction. In this study cysteine mutagenesis and oxidative cross-linking were combined to analyze proximity relationships of helices 1, 2, and 7 in LdNT1.1. Disulfide bond formation between introduced paired cysteines at the interface of such helices (A61CTM1/F74CTM2, A61CTM1/G350C TM7, and F74CTM2/G350CTM7) was analyzed by transport measurement and gel mobility shifts upon oxidation with Cu (II)-(1,10-phenanthroline)3. In all cases cross-linking inhibited transport. However, if LdNT1.1 ligands were included during cross-linking, inhibition of transport was reduced, suggesting that ligands moved the three gating helices apart. Moreover, all paired cysteine mutants exhibited a mobility shift upon oxidation, corroborating the formation of a disulfide bond. These data support the notion that helices 1, 2, and 7 constitute the extracellular gate of LdNT1.1, thus further validating the computational model and the previously demonstrated importance of F48TM1 and Trp-75TM2 in tethering together helices that are part of the gate.

Original languageEnglish (US)
Pages (from-to)44036-44045
Number of pages10
JournalJournal of Biological Chemistry
Volume287
Issue number53
DOIs
StatePublished - Dec 28 2012

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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