Abstract
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophic. They import purines via an equilibrative nucleoside transporter (ENT). In P. falciparum, the most virulent species, the equilibrative nucleoside transporter 1 (PfENT1) represents the primary purine uptake pathway. This transporter is a potential target for the development of antimalarial drugs. In the absence of a high-resolution structure for either PfENT1 or a homologous ENT, we used the substituted cysteine accessibility method (SCAM) to investigate the membrane-spanning domain structure of PfENT1 to identify potential inhibitor-binding sites. We previously used SCAM to identify water-accessible residues that line the permeation pathway in transmembrane segment 11 (TM11). TM2 and TM10 lie adjacent to TM11 in an ab initio model of a homologous Leishmania donovani nucleoside transporter. To identify TM2 and TM10 residues in PfENT1 that are at least transiently on the water-accessible transporter surface, we assayed the reactivity of single cysteine-substitution mutants with three methanethiosulfonate (MTS) derivatives. Cysteines substituted for 12 of 14 TM2 segment residues reacted with MTS-ethyl-ammonium-biotin (MTSEA-biotin). At eight positions, MTSEA-biotin inhibited transport, and at four positions substrate transport was potentiated. On an α helical wheel projection of TM2, the four positions where potentiation occurred were located in a cluster on one side of the helix. In contrast, although MTSEA-biotin inhibited 9 of 10 TM10 cysteine-substituted mutants, the reactive residues did not form a pattern consistent with either an α helix or β sheet. These results may help identify the binding site(s) of PfENT1 inhibitors.
Highlights
Infection with Plasmodium species parasites causes malaria
Our results indicate that Cys substituted for some TM2 residues reacted with the MTS reagents
We assayed the effect of a 5-min application of increasing concentrations of the three MTS reagents on [3H]adenosine ([3H]Ado) uptake into yeast expressing WT PfENT1
Summary
Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophic. In P. falciparum, the most virulent species, the equilibrative nucleoside transporter 1 (PfENT1) represents the primary purine uptake pathway. This transporter is a potential target for the development of antimalarial drugs. We previously used SCAM to identify water-accessible residues that line the permeation pathway in transmembrane segment 11 (TM11). To identify TM2 and TM10 residues in PfENT1 that are at least transiently on the water-accessible transporter surface, we assayed the reactivity of single cysteine-substitution mutants with three methanethiosulfonate (MTS) derivatives. Our results indicate that Cys substituted for some TM2 residues reacted with the MTS reagents We infer that they are water accessible and may line the permeation pathway. The pattern formed by the TM10 reactive residues was not consistent with either an ␣ helix or  sheet
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