Accessibility of substituted cysteines in TM2 and TM10 transmembrane segments in the Plasmodium falciparum equilibrative nucleoside transporter PfENT1

Sita Nirupama Nishtala, Avish Arora, Jorge Reyes, Myles Akabas

Research output: Contribution to journalArticle

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 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 helix or sheet. These results may help identify the binding site(s) of PfENT1 inhibitors.

Original languageEnglish (US)
Pages (from-to)1924-1935
Number of pages12
JournalJournal of Biological Chemistry
Volume294
Issue number6
DOIs
StatePublished - Jan 1 2019

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Equilibrative Nucleoside Transporter 1
Nucleoside Transport Proteins
Plasmodium falciparum
Cysteine
Biotin
Ammonium Compounds
Malaria
Parasites
Binding Sites
Leishmania donovani
Purines
Plasmodium
Water
Antimalarials
Permeation
Wheels
Substitution reactions
Derivatives
Membranes
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Accessibility of substituted cysteines in TM2 and TM10 transmembrane segments in the Plasmodium falciparum equilibrative nucleoside transporter PfENT1. / Nishtala, Sita Nirupama; Arora, Avish; Reyes, Jorge; Akabas, Myles.

In: Journal of Biological Chemistry, Vol. 294, No. 6, 01.01.2019, p. 1924-1935.

Research output: Contribution to journalArticle

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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 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 helix or sheet. These results may help identify the binding site(s) of PfENT1 inhibitors.",
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