Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.

PARP7 mono-ADP-ribosylates the agonist conformation of the androgen receptor in the nucleus.

We recently described a signal transduction pathway that contributes to androgen receptor (AR) regulation based on site-specific ADP-ribosylation by PARP7, a mono-ADP-ribosyltransferase implicated in several human cancers. ADP-ribosylated AR is recognized by PARP9/DTX3L, a heterodimeric complex that contains an ADP-ribose reader (PARP9) and a ubiquitin E3 ligase (DTX3L). Here, we have characterized the cellular and biochemical requirements for AR ADP-ribosylation by PARP7. We found that the reaction requires nuclear localization of PARP7 and an agonist-induced conformation of AR. PARP7 contains a Cys3His1-type zinc finger (ZF), which also is critical for AR ADP-ribosylation. The Parp7 ZF is required for efficient nuclear import by a nuclear localization signal encoded in PARP7, but rescue experiments indicate the ZF makes a contribution to AR ADP-ribosylation that is separable from the effect on nuclear transport. ZF mutations do not detectably reduce PARP7 catalytic activity and binding to AR, but they do result in the loss of PARP7 enhancement of AR-dependent transcription of the MYBPC1 gene. Our data reveals critical roles for AR conformation and the PARP7 ZF in AR ADP-ribosylation and AR-dependent transcription.

Chemical genetics and proteome-wide site mapping reveal cysteine MARylation by PARP-7 on immune-relevant protein targets.

Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13-a critical regulator of the antiviral innate immune response-is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.

Toward a Molecular Basis of Cellular Nucleoside Transport in Humans.

Nucleosides play central roles in all facets of life, from metabolism to cellular signaling. Because of their physiochemical properties, nucleosides are lipid bilayer impermeable and thus rely on dedicated transport systems to cross biological membranes. In humans, two unrelated protein families mediate nucleoside membrane transport: the concentrative and equilibrative nucleoside transporter families. The objective of this review is to provide a broad outlook on the current status of nucleoside transport research. We will discuss the role played by nucleoside transporters in human health and disease, with emphasis placed on recent structural advancements that have revealed detailed molecular principles of these important cellular transport systems and exploitable pharmacological features.

HPLC reveals novel features of nucleoside and nucleobase homeostasis, nucleoside metabolism and nucleoside transport.

Humans possess three members of the cation-coupled concentrative nucleoside transporter CNT (SLC 28) family, hCNT1-3: hCNT1 is selective for pyrimidine nucleosides but also transports adenosine, hCNT2 transports purine nucleosides and uridine, and hCNT3 transports both pyrimidine and purine nucleosides. hCNT1/2 transport nucleosides using the transmembrane Na+ electrochemical gradient, while hCNT3 is both Na+- and H+-coupled. By producing recombinant hCNT3 in Xenopus laevis oocytes, we have used radiochemical high performance liquid chromatography (HPLC) analysis to investigate the metabolic fate of transported [3H] or [14C] pyrimidine and purine nucleosides once inside cells. With the exception of adenosine, transported nucleosides were generally subject to minimal intracellular metabolism. We also used radiochemical HPLC analysis to study the mechanism by which adenosine functions as a low Km, low Vmax permeant of hCNT1. hCNT1-producing oocytes were pre-loaded with [3H] uridine, after which efflux of accumulated radioactivity was measured in transport medium alone, or in the presence of extracellular non-radiolabelled adenosine or uridine. hCNT1-mediated [3H]-efflux was stimulated by extracellular uridine, but inhibited by extracellular adenosine, with >95% of the radioactivity exiting cells being unmetabolized uridine, consistent with a low transmembrane mobility of the hCNT1/adenosine complex. Humans also possess four members of the equilibrative nucleoside transporter ENT (SLC 29) family, hENT1-4. Of these, hENT1 and hENT2 transport both nucleosides and nucleobases into and out of cells, but their relative contributions to nucleoside and nucleobase homeostasis and, in particular, to adenosine signaling via purinoreceptors, are not known. We therefore used HPLC to determine plasma nucleoside and nucleobase concentrations in wild-type, mENT1-, mENT2- and mENT1/mENT2-knockout (KO) mice, and to compare the findings with knockout of mCNT3. Results demonstrated that ENT1 was more important than ENT2 or CNT3 in determining plasma adenosine concentrations, indicated modest roles of ENT1 in the homeostasis of other nucleosides, and suggested that none of the transporters is a major participant in handling of nucleobases.

Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family.

Borrelia burgdorferisensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato Because B. burgdorferisensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

Characterization of drug resistance-associated TevAT1 gene of Trypanosoma evansi from Philippine water buffaloes (Bubalus bubalis)

This study detected and characterized the TevAT1 gene of Trypanosoma evansi isolates from Philippine water buffaloes (Bubalus bubalis). A total of 68 blood samples from Philippine water buffaloes were subjected to DNA extraction and PCR assay was performed using RoTat 1.2 gene to detect T. evansi. Those samples positive for T. evansi subsequently underwent another PCR assay to detect the presence of TevAT1 gene. Trypanosoma evansi was detected in 26.47% (18/68) blood samples in which distributed throughout the main islands of the country (4 from Luzon, 2 from Visayas and 12 from Mindanao). However, only 10 of these samples were positive for TevAT1 gene. Sequence alignment of the TevAT1 gene from local isolates showed no single nucleotide polymorphisms when compared to other strains in various countries. Those T. evansi without the gene of interest could be possibly resistant to some trypanocidal drugs but this needs to be further investigated in-vitro or in-vivo.

Sclerosing bone dysplasias with hallmarks of dysosteosclerosis in four patients carrying mutations in SLC29A3 and TCIRG1.

The osteopetroses and related sclerosing bone dysplasias can have a broad range of manifestations. Especially in the milder forms, sandwich vertebrae are an easily recognizable and reliable radiological hallmark. We report on four patients from three families presenting with sandwich vertebrae and platyspondyly. The long bone phenotypes were discordant with one patient showing modeling defects and patchy osteosclerosis, while the second displayed only metaphyseal sclerotic bands, and the third and fourth had extreme metaphyseal flaring with uniform osteosclerosis. Two of the four patients had experienced pathological fractures, two had developmental delay, but none showed cranial nerve damage, hepatosplenomegaly, or bone marrow failure. According to these clinical features the diagnoses ranged between intermediate autosomal recessive osteopetrosis and dysosteosclerosis. After exclusion of mutations in CLCN7 we performed gene panel and exome sequencing. Two novel mutations in SLC29A3 were found in the first two patients. In the third family a TCIRG1 C-terminal frameshift mutation in combination with a mutation at position +4 in intron 2 were detected. Our study adds two cases to the small group of individuals with SLC29A3 mutations diagnosed with dysosteosclerosis, and expands the phenotypic variability. The finding that intermediate autosomal recessive osteopetrosis due to TCIRG1 splice site mutations can also present with platyspondyly further increases the molecular heterogeneity of dysosteosclerosis-like sclerosing bone dysplasias.

Fluorinated nucleosides as an important class of anticancer and antiviral agents.

Fluorine-containing nucleoside analogs (NAs) represent a significant class of the US FDA-approved chemotherapeutics widely used in the clinic. The incorporation of fluorine into drug-like agents modulates lipophilic, electronic and steric parameters, thus influencing pharmacodynamic and pharmacokinetic properties of drugs. Fluorine can block oxidative metabolism of drugs and the formation of undesired metabolites by changing H-bonding interactions. In this review, we focus our attention on chemical fluorination reagents and methods used in the NAs field, including positron emission tomography radiochemistry. We briefly discuss both the cellular biology and clinical properties of FDA-approved and fluorine-containing nucleoside/nucleotide analogs in development as well as common resistance mechanisms associated with their use. Finally, we emphasize pronucleotide strategies used to improve therapeutic outcome of NAs in the clinic.

Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3.

Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5-7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity.

Visualizing multistep elevator-like transitions of a nucleoside transporter.

Membrane transporters move substrates across the membrane by alternating access of their binding sites between the opposite sides of the membrane. An emerging model of this process is the elevator mechanism, in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states, but the conformational path that leads to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of the inward-facing, intermediate, and outward-facing states of a concentrative nucleoside transporter from Neisseria wadsworthii. Notably, we determined the structures of multiple intermediate conformations, in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain that are associated with the elevator-like motion.

Equilibrative nucleoside transporters-A review.

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that mediate the transport of nucleosides, nucleobases, and therapeutic analogs. The best-characterized ENTs are the human transporters hENT1 and hENT2. However, non-mammalian eukaryotic ENTs have also been studied (e.g., yeast, parasitic protozoa). ENTs are major pharmaceutical targets responsible for modulating the efficacy of more than 30 approved drugs. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. This review highlights findings on the characterization of ENTs by surveying studies on genetics, permeant and inhibitor interactions, mutagenesis, and structural models of ENT function.

A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.

Functional analysis of drug resistance-associated mutations in the Trypanosoma brucei adenosine transporter 1 (TbAT1) and the proposal of a structural model for the protein.

The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.

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

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.

Identification of the intracellular gate for a member of the equilibrative nucleoside transporter (ENT) family.

Equilibrative nucleoside transporters of the SLC29 family play important roles in many physiological and pharmacological processes, including import of drugs for treatment of cancer, AIDS, cardiovascular, and parasitic diseases. However, no crystal structure is available for any member of this family. In previous studies we generated a computational model of the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1) that captured this permease in the outward-closed conformation, and we identified the extracellular gate. In the present study we have modeled the inward-closed conformation of LdNT1.1 using the crystal structure of the Escherichia coli fucose transporter FucP and have identified four transmembrane helices whose ends close to form a predicted intracellular gate. We have tested this prediction by site-directed mutagenesis of relevant helix residues and by cross-linking of introduced cysteine pairs. The results are consistent with the predictions of the computational model and suggest that a similarly constituted gate operates in other members of the equilibrative nucleoside transporter family.

Functional identification of the hypoxanthine/guanine transporters YjcD and YgfQ and the adenine transporters PurP and YicO of Escherichia coli K-12.

The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N(6)-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.

Crithidia fasciculata adenosine transporter 1 (CfAT1), a novel high-affinity equilibrative nucleoside transporter specific for adenosine.

Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower Km for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.

Use of molecular modelling to probe the mechanism of the nucleoside transporter NupG.

Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.

Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å.

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.