Transmembrane Domain 5 of the LdNT1.1 Nucleoside Transporter Is an Amphipathic Helix That Forms Part of the Nucleoside Translocation Pathway

Abstract

Transporters of the equilibrative nucleoside transporter (ENT) family promote the uptake of nucleosides, nucleobases, and a variety of therapeutic drugs in eukaryotes from protozoa to mammals. Despite its importance, the translocation pathway that mediates the internalization of these substrates has not been identified yet in any of the ENT carriers. Previous genetic studies on the LdNT1.1 nucleoside transporter from Leishmania donoVani defined two amino acid residues in predicted transmembrane domains (TMD) 5 and 7 that may line this translocation pathway. The role of TMD5 in forming a portion of the aqueous channel was investigated using the substituted-cysteine accessibility method. A series of 22 cysteine substitution mutants spanning predicted TMD5 were created from a fully functional, cysteine-less, parental LdNT1.1. Cysteine replacement at six positions (M 176 C, T 186 C, S 187 C, Q 190 C, V 193 C, and K 194 C) produced permeases that were inhibited by incubation with sulfhydryl-specific methanethiosulfonate reagents, denoting their solvent accessibility to the translocation pathway. Adenosine was able to block this thiol modification, implying that access to the domain becomes restricted as a consequence of the substrate binding. Strikingly, the Q 190 C substitution interacted differentially with the substrates adenosine and uridine, suggesting that binding of adenosine but not uridine might directly occlude this position. When superimposed on a helical model, all six mutants clustered along one face of the amphipathic R-helix predicted for TMD5, strongly suggesting its involvement in the translocation pathway through LdNT1.1.Leishmania donoVani is the causative agent of visceral leishmaniasis, a devastating and often fatal disease if untreated. The parasite exhibits a digenetic lifecycle in which the extracellular, flagellated, and motile promastigote resides within the phlebotomine sandfly vector, and the intracellular, aflagellar, and nonmotile amastigote exists within the phagolysosome of macrophages and other reticuloendothelial cells of the mammalian host. Purine salvage pathways are critical to the survival of parasitic protozoa, because all these organisms lack the ability to synthesize the purine ring de novo (1). This metabolic disparity between parasites and their mammalian hosts, which do synthesize purines, has been exploited for rational development of improved therapies and for design of selective antiparasitic drugs (2). Since the initial step in purine salvage involves the translocation of preformed host purines across the parasite surface membranes, parasite nucleoside and nucleobase transporters may constitute potential targets for chemotherapy or routes for delivery of purine analogue drugs.The LdNT1 transporters of L. donoVani mediate the uptake of adenosine, pyrimidine nucleosides, and the cytotoxic adenosine analogue tubercidin. The LdNT1 genetic locus encompasses two closely related genes, LdNT1.1 and LdNT1.2, which were cloned by functional complementation (3) of an adenosine/pyri...

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

“…The magenta rectangle on TM5 denotes the face of TM5 in LdNT1 that was found to be solvent-accessible (27). The roles of these residues in transport function and ligand recognition are described under "Discussion."…”

“…7A) is supported by the location of key residues within TMs 1, 2, 4, and 5, all of which map toward the putative aqueous pore in the model, that have been identified by structure-function studies to be critical for ligand selectivity or to be solvent-accessible (4 -6, 8, 27, 28, 36). Specifically these include the following: Met 33 and Leu 92 within hENT1, which have been implicated in the interaction with the vasodilators, dipyridamole and dilazep (Met 33 (5) and LdNT1 (4), respectively, that are important determinants for uridine transport as well as for dilazep interaction in the case of Gly 179 ; Asp 389 and Arg 393 within TM8 of LdNT2, which constitute a conserved signature motif (DXXXR) within the majority of ENTs (10) and are important determinants for transporter function and targeting (11); Cys 337 in LdNT1,which when mutated to a bulkier residue results in a transporter with impaired transport capability (4); residues mapped to the solvent-accessible face of TM5 in LdNT1 (27); and an exofacial Cys 140 in rENT1 (28). Because of the low sequence identity (Ͻ14%) between the MFS and ENTs and the inherent difficulties in modeling unstructured loop domains, this ENT structural prediction will, of course, require extensive computational refinement and experimental validation to evolve.…”

“…7A). Furthermore, all the residues on various ENTs that have been identified through biochemical and genetic data to be either solvent-accessible (27,28) or important ligand selectivity determinants (4 -8) were oriented toward the putative ligand-binding pore (Fig. 7A).…”

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

“…The tag was cloned into the BglII site of the Leishmania pX63NEORI expression vector (22). Subsequently, the open reading frame of LmxGT1 was amplified and cloned into the BamHI and EcoRI sites to generate the LmxGT1::HBH gene fusion.…”

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

“…This generally requires that most or all naturally occurring cysteine residues in the protein be replaced with other amino acids without altering the structure of the protein, as assessed by comparing function to its wild-type counterpart. Mutation of cysteine residues to alanine or serine in many cases results in a cysteine-less protein retaining wild-type affinity for ligand (see [9][10][11]); however, in other proteins such substitutions result in a non-functional protein [12][13][14]. Identification of substitutions that are functional may not be straightforward in such cases, requiring a significant number of molecular biological and biochemical manipulations to construct and evaluate mutants that one thinks might be functional.…”