Putative purine nucleoside interacting residues in the malaria parasite purine uptake transporter PfENT1 are critical for transporter function.

Malaria remains a major public health threat for billions of people worldwide. Infection with obligate intracellular, unicellular parasites from the genus Plasmodium causes malaria. Plasmodium falciparum causes the deadliest form of human malaria. Plasmodium parasites are purine auxotrophic. They rely on purine import from the host red blood cell cytoplasm via equilibrative nucleoside transporters to supply substrates to the purine salvage pathway. We previously developed a high throughput screening assay to identify inhibitors of the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Screening a small molecule library identified PfENT1 inhibitors that blocked proliferation of P. falciparum parasites in in vitro culture. The goal of the current work was to validate a high-resolution model of PfENT1 predicted by the AlphaFold protein structure prediction program. We superimposed the predicted PfENT1 structure on the human homologue structure, hENT1, and developed a structure-based sequence alignment. We mutated the residues in PfENT1 aligned with and flanking the residues in hENT1 that interact with the purine analog, nitrobenzylthioinosine (NBMPR). Mutation of the PfENT1 residues Q135, D287, and R291 that are predicted to form hydrogen bonds to purine nucleosides eliminated purine and pyrimidine transport function in various yeast-based growth and radiolabeled substrate uptake assays. Mutation of two flanking residues, W53 and S290, also resulted in inactive protein. Mutation of L50 that forms hydrophobic interactions with the purine nucleobase reduced transport function. Based on our results the AlphaFold predicted structure for PfENT1 may be useful in guiding medicinal chemistry efforts to improve the potency of our PfENT1 inhibitors.

Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1.

By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.

Impact of Field Isolate Identified Nonsynonymous Single Nucleotide Polymorphisms on Plasmodium falciparum Equilibrative Nucleoside Transporter 1 Inhibitor Efficacy.

Plasmodium falciparum causes the most severe form of malaria and causes approximately 500 000 deaths per year. P. falciparum parasites resistant to current antimalarial treatments are spreading. Therefore, it is imperative to develop new antimalarial drugs. Malaria parasites are purine auxotrophic. They rely on purine import from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Recently, inhibitors of the P. falciparum ENT1 (PfENT1) that inhibit proliferation of malaria parasites in culture have been identified as promising starting points for antimalarial drug development. Genome sequencing of P. falciparum field isolates has identified nonsynonymous single nucleotide polymorphisms (SNPs) in the gene encoding PfENT1. Here we evaluate the impact of these PfENT1 SNPs on purine substrate affinity and inhibitor efficacy. We expressed each PfENT1-SNP in Saccharomyces cerevisiae. Using PfENT1-SNP-expressing yeast, we characterized the PfENT1 purine substrate affinity using radiolabeled substrate uptake inhibition experiments. Four of the 13 SNPs altered affinity for one or more purines by up to 7-fold. Three of the SNPs reduced the potency of a subset of the inhibitors by up to 7-fold. One SNP, Q284E, reduced the potency of all six inhibitor chemotypes. We tested drug efficacy in available parasite strains containing PfENT1 SNPs. While PfENT1-SNP-expressing yeast had decreased sensitivity to PfENT1 inhibitors, parasite strains containing SNPs showed similar or more potent inhibition of proliferation with all PfENT1 inhibitors. Thus, parasite strains bearing PfENT1 SNPs are not resistant to these PfENT1 inhibitors. This supports PfENT1 as a promising target for further development of novel antimalarial drugs.

Identification via a Parallel Hit Progression Strategy of Improved Small Molecule Inhibitors of the Malaria Purine Uptake Transporter that Inhibit Plasmodium falciparum Parasite Proliferation.

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and ∼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.

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

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

NmeA, a novel efflux transporter specific for nucleobases and nucleosides, contributes to metal resistance in Aspergillus nidulans.

Through Minos transposon mutagenesis we obtained A. nidulans mutants resistant to 5-fluorouracil due to insertions into the upstream region of the uncharacterized gene nmeA, encoding a Major Facilitator Superfamily (MFS) transporter. Minos transpositions increased nmeA transcription, which is otherwise extremely low under all conditions tested. To dissect the function of NmeA we used strains overexpressing or genetically lacking the nmeA gene. Strains overexpressing NmeA are resistant to toxic purine analogues, but also, to cadmium, zinc and borate, whereas an isogenic nmeAΔ null mutant exhibits increased sensitivity to these compounds. We provide direct evidence that nmeA overexpression leads to efflux of adenine, xanthine, uric acid and allantoin, the latter two being intermediate metabolites of purine catabolism that are toxic when accumulated cytoplasmically due to relevant genetic lesions. By using a functional GFP-tagged version we show that NmeA is a plasma membrane transporter. Homology modeling and docking approaches identified a single purine binding site and a tentative substrate translocation trajectory in NmeA. Orthologues of NmeA are present in all Aspergilli and other Eurotiomycetes, but are absent from other fungi or non-fungal organisms. NmeA is thus the founding member of a new class of transporters essential for fungal success under specific toxic conditions.

Molecular mechanisms of substrate-controlled ring dynamics and substepping in a nucleic acid-dependent hexameric motor.

Ring-shaped hexameric helicases and translocases support essential DNA-, RNA-, and protein-dependent transactions in all cells and many viruses. How such systems coordinate ATPase activity between multiple subunits to power conformational changes that drive the engagement and movement of client substrates is a fundamental question. Using the Escherichia coli Rho transcription termination factor as a model system, we have used solution and crystallographic structural methods to delineate the range of conformational changes that accompany distinct substrate and nucleotide cofactor binding events. Small-angle X-ray scattering data show that Rho preferentially adopts an open-ring state in solution and that RNA and ATP are both required to cooperatively promote ring closure. Multiple closed-ring structures with different RNA substrates and nucleotide occupancies capture distinct catalytic intermediates accessed during translocation. Our data reveal how RNA-induced ring closure templates a sequential ATP-hydrolysis mechanism, provide a molecular rationale for how the Rho ATPase domains distinguishes between distinct RNA sequences, and establish structural snapshots of substepping events in a hexameric helicase/translocase.

Targeting the Plasmodium vivax equilibrative nucleoside transporter 1 (PvENT1) for antimalarial drug development.

Infection with Plasmodium falciparum and vivax cause most cases of malaria. Emerging resistance to current antimalarial medications makes new drug development imperative. Ideally a new antimalarial drug should treat both falciparum and vivax malaria. Because malaria parasites are purine auxotrophic, they rely on purines imported from the host erythrocyte via Equilibrative Nucleoside Transporters (ENTs). Thus, the purine import transporters represent a potential target for antimalarial drug development. For falciparum parasites the primary purine transporter is the P. falciparum Equilibrative Nucleoside Transporter Type 1 (PfENT1). Recently we identified potent PfENT1 inhibitors with nanomolar IC50 values using a robust, yeast-based high throughput screening assay. In the current work we characterized the Plasmodium vivax ENT1 (PvENT1) homologue and its sensitivity to the PfENT1 inhibitors. We expressed a yeast codon-optimized PvENT1 gene in Saccharomyces cerevisiae. PvENT1-expressing yeast imported both purines ([(3)H]adenosine) and pyrimidines ([(3)H]uridine), whereas wild type (fui1Δ) yeast did not. Based on radiolabel substrate uptake inhibition experiments, inosine had the lowest IC50 (3.8 µM), compared to guanosine (14.9 µM) and adenosine (142 µM). For pyrimidines, thymidine had an IC50 of 183 µM (vs. cytidine and uridine; mM range). IC50 values were higher for nucleobases compared to the corresponding nucleosides; hypoxanthine had a 25-fold higher IC50 than inosine. The archetypal human ENT1 inhibitor 4-nitrobenzylthioinosine (NBMPR) had no effect on PvENT1, whereas dipyridamole inhibited PvENT1, albeit with a 40 µM IC50, a 1000-fold less sensitive than human ENT1 (hENT1). The PfENT1 inhibitors blocked transport activity of PvENT1 and the five known naturally occurring non-synonymous single nucleotide polymorphisms (SNPs) with similar IC50 values. Thus, the PfENT1 inhibitors also target PvENT1. This implies that development of novel antimalarial drugs that target both falciparum and vivax ENT1 may be feasible.

Yeast-based high-throughput screen identifies Plasmodium falciparum equilibrative nucleoside transporter 1 inhibitors that kill malaria parasites.

Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite Plasmodium falciparum (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64 560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing fui1Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC50 0.2-2 µM). These nine compounds completely blocked [(3)H]adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC50 5-50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC50 5-50 µM). Wild-type (WT) parasite IC50 values were up to 4-fold lower compared to PfENT1-knockout (pfent1Δ) parasites. pfent1Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development.

Purine import into malaria parasites as a target for antimalarial drug development.

Infection with Plasmodium species parasites causes malaria. Plasmodium parasites are purine auxotrophs. In all life cycle stages, they require purines for RNA and DNA synthesis and other cellular metabolic processes. Purines are imported from the host erythrocyte by equilibrative nucleoside transporters (ENTs). They are processed via purine salvage pathway enzymes to form the required purine nucleotides. The Plasmodium falciparum genome encodes four putative ENTs (PfENT1-4). Genetic, biochemical, and physiologic evidence suggest that PfENT1 is the primary purine transporter supplying the purine salvage pathway. Protein mass spectrometry shows that PfENT1 is expressed in all parasite stages. PfENT1 knockout parasites are not viable in culture at purine concentrations found in human blood (<10 µM). Thus, PfENT1 is a potential target for novel antimalarial drugs, but no PfENT1 inhibitors have been identified to test the hypothesis. Identifying inhibitors of PfENT1 is an essential step to validate PfENT1 as a potential antimalarial drug target.

Loading strategies of ring-shaped nucleic acid translocases and helicases.

Ring-shaped nucleic acid translocases and helicases catalyze the directed and processive movement of nucleic acid strands to support essential transactions such as replication, transcription, and chromosome partitioning. Assembled typically as hexamers, ring helicase/translocase systems use coordinated cycles of nucleoside triphosphate (NTP) hydrolysis to translocate extended DNA or RNA substrates through a central pore. Ring formation presents a topological challenge to the engagement of substrate oligonucleotides, and is frequently overcome by distinct loading strategies for shepherding specific motors onto their respective substrates. Recent structural studies that capture different loading intermediates have begun to reveal how different helicase/translocase rings either assemble around substrates or crack open to allow DNA or RNA strand entry, and how dedicated chaperones facilitate these events in some instances. Both prevailing mechanistic models and remaining knowledge gaps are discussed.

An epidermal microRNA regulates neuronal migration through control of the cellular glycosylation state.

An appropriate balance in glycosylation of proteoglycans is crucial for their ability to regulate animal development. Here, we report that the Caenorhabditis elegans microRNA mir-79, an ortholog of mammalian miR-9, controls sugar-chain homeostasis by targeting two proteins in the proteoglycan biosynthetic pathway: a chondroitin synthase (SQV-5; squashed vulva-5) and a uridine 5'-diphosphate-sugar transporter (SQV-7). Loss of mir-79 causes neurodevelopmental defects through SQV-5 and SQV-7 dysregulation in the epidermis. This results in a partial shutdown of heparan sulfate biosynthesis that impinges on a LON-2/glypican pathway and disrupts neuronal migration. Our results identify a regulatory axis controlled by a conserved microRNA that maintains proteoglycan homeostasis in cells.

The DNA uptake ATPase PilF of Thermus thermophilus: a reexamination of the zinc content.

The DNA-translocator ATPase PilF of Thermus thermophilus HB27 is a hexamer built by six identical subunits. Despite the presence of a conserved zinc-binding site in every subunit, only one zinc atom per hexamer was found. Re-examination of the zinc content of PilF purified from cells grown in complex media with different lots of yeast extract revealed six zinc atoms per hexamer. These data demonstrate that the low zinc content reported before was most likely a result of zinc depletion of the yeast extract used.

Disruption of lmo1386, a putative DNA translocase gene, affects biofilm formation of Listeria monocytogenes on abiotic surfaces.

The distribution and survival of Listeria monocytogenes (L. monocytogenes) in food processing environment is linked to its ability to form biofilms, however the genetic mechanisms remain unclear. In our previous study, a Himar1 mariner-based transposon mutagenesis was performed and 42 mutants were confirmed to have reduced biofilm formation. Among the 42 biofilm deficient mutants, two isolates (s25-10C and s55-1D) harbored single insertion in lmo1386, a gene encoding a putative DNA translocase. The lmo1386 mutants had impaired biofilm formation in both static and flow conditions. The mutant strain s55-1D was complemented by cloning the entire lmo1386 gene into pPL2-gtcAP, a derivative of the integration vector pPL2 with the L. monocytogenes gtcA promoter. The genetically complemented mutant restored its biofilm phenotype, demonstrating the role of lmo1386 in the biofilm formation of L. monocytogenes. The lmo1386 mutant had reduced initial adhesion ability, which could at least partially contribute to the impaired biofilm phenotype. Additionally, the lmo1386 mutant formed elongated cell chains when grown in a nutrient TSBYE media, while no obvious cell morphology changes were observed when grown in the minimal MWB media. Overall, our findings suggest that the disruption of lmo1386, a putative DNA translocase gene affects the biofilm formation of L. monocytogenes on abiotic surfaces, which may further advance the understanding of the complicated process of biofilm formation.

Nucleoside transporters: biological insights and therapeutic applications.

Nucleoside transporters play important physiological roles by regulating intra- and extra-cellular concentrations of purine and pyrimidine (deoxy)nucleosides. This review describes the biological function and activity of the two major families of membrane nucleoside transporters that exist in mammalian cells. These include equilibrative nucleoside transporters that transport nucleosides in a gradient-dependent fashion and concentrative nucleoside transporters that import nucleosides against a gradient by coupling movement with sodium transport. Particular emphasis is placed on describing the roles of nucleoside transport in normal physiological processes, including inflammation, cardiovascular function and nutrient transport across the blood-brain barrier. In addition, the role of nucleoside transport in pathological conditions such as cardiovascular disease and cancer are discussed. The potential therapeutic applications of manipulating nucleoside transport activities are discussed, focusing on nucleoside analogs as anti-neoplastic agents. Finally, we discuss future directions for the development of novel chemical entities to measure nucleoside transport activity at the cellular and organismal level.

A protein shuttle system to target RNA into mitochondria.

Mitochondria play a key role in essential cellular functions. A deeper understanding of mitochondrial molecular processes is hampered by the difficulty of incorporating foreign nucleic acids into organelles. Mitochondria of most eukaryotic species import cytosolic tRNAs. Based on this natural process, we describe here a powerful shuttle system to internalize several types of RNAs into isolated mitochondria. We demonstrate that this tool is useful to investigate tRNA processing or mRNA editing in plant mitochondria. Furthermore, we show that the same strategy can be used to address both tRNA and mRNA to isolated mammalian mitochondria. We anticipate our novel approach to be the starting point for various studies on mitochondrial processes. Finally, our study provides new insights into the mechanism of RNA import into mitochondria.

Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1.

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the K(m) and/or K(i) for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the K(i) for PfENT1 transport of hypoxanthine and adenosine is in the 300-700microM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport K(m). PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [(3)H]hypoxanthine or [(3)H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

Plasmodium falciparum var gene expression is modified by host immunity.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, which play a central role in the host-parasite interaction by binding to various host molecules. They are encoded by a diverse family of genes called var, of which there are approximately 60 copies in each parasite genome. In sub-Saharan Africa, although P. falciparum infection occurs throughout life, severe malarial disease tends to occur only in childhood. This could potentially be explained if (i) PfEMP1 variants differ in their capacity to support pathogenesis of severe malaria and (ii) this capacity is linked to the likelihood of each molecule being recognized and cleared by naturally acquired antibodies. Here, in a study of 217 Kenyan children with malaria, we show that expression of a group of var genes "cys2," containing a distinct pattern of cysteine residues, is associated with low host immunity. Expression of cys2 genes was associated with parasites from young children, those with severe malaria, and those with a poorly developed antibody response to parasite-infected erythrocyte surface antigens. Cys-2 var genes form a minor component of all genomic var repertoires analyzed to date. Therefore, the results are compatible with the hypothesis that the genomic var gene repertoire is organized such that PfEMP1 molecules that confer the most virulence to the parasite tend also to be those that are most susceptible to the development of host immunity. This may help the parasite to adapt effectively to the development of host antibodies through modification of the host-parasite relationship.

A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica.

Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix-turn-helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix alpha1 contacts tRNA whereas helix alpha2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.