An ab Initio Structural Model of a Nucleoside Permease Predicts Functionally Important Residues

Abstract

Permeases belonging to the equilibrative nucleoside transporter family promote uptake of nucleosides and/or nucleobases into a wide range of eukaryotes and mediate the uptake of a variety of drugs used in the treatment of cancer, heart disease, AIDS, and parasitic infections. No experimental three-dimensional structure exists for any of these permeases, and they are not present in prokaryotes, the source of many membrane proteins used in crystal structure determination. To generate a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani was modeled using ab initio computation. Site-directed mutations that strongly impair transport or that alter substrate specificity map to the central pore of the ab initio model, whereas mutations that have less pronounced phenotypes map to peripheral positions. The model suggests that aromatic residues present in transmembrane helices 1, 2, and 7 may interact to form an extracellular gate that closes the permeation pathway in the inward oriented conformation. Mutation of two of these three residues abrogated transport activity, consistent with the prediction of the model. The ab initio model is similar to one derived previously using threading analysis, a distinct computational approach, supporting the overall accuracy of both models. However, significant differences in helix orientation and residue position between the two models are apparent, and the mutagenesis data suggest that the ab initio model represents an improvement regarding structural details over the threading model. The putative gating interaction may also help explain differences in substrate specificity between members of this family.Nucleoside transporters play pivotal roles in nucleoside salvage pathways, regulation of adenosine signaling, and the pharmacology of antineoplastic and antiviral nucleoside drugs (1, 2).Salvage of nucleosides and nucleobases is the first step of nucleoside utilization in those cells that lack the metabolic machinery to make purine nucleotides de novo, including protozoan parasites (3) and brain and bone marrow cells in mammals (4). Nucleoside permeases also mediate the uptake of a number of nucleoside analog drugs used to combat the devastating effects of chronic diseases, including those caused by RNA viruses, cancer, and parasitic protozoan infections (5, 6).Equilibrative nucleoside transporters (ENTs) 4 are a unique family of proteins (the SLC29 family), with no apparent sequence homology to other types of permeases, that enable facilitated diffusion of nucleosides, nucleoside analogs, and nucleobases across cell membranes. Although widely distributed among eukaryotes from protozoa to humans, ENT-like homologs have not been identified in prokaryotes, and therefore crystallization of these transporters is likely to be even more challenging than for those membrane proteins that do have orthologs in prokaryotes. In the absence of a crystallographic structure, the use of genetic and biochemical approaches, especial...

Identification of the Intracellular Gate for a Member of the Equilibrative Nucleoside Transporter (ENT) Family

“…The ab initio model predicted that most of the LdNT1.1 mutants that we previously found to greatly impair transport activity or alter substrate specificity (15,18) line the central permeation pore, as might be expected for mutations that induce strong transport phenotypes. In addition, analogous to the experimentally determined structures for the bacterial permeases LacY and GlpT, the LdNT1.1 ab initio model predicted that TM helices 1, 2, and 7 cluster together at the extracellular surface of the transporter to close off the pore or permeation pathway and form an "extracellular gate" (15,19). Two lines of experimental evidence support the identification of this extracellular gate.…”

“…Homology Modeling-Because the previous computational model of LdNT1.1 captured the permease in an outwardclosed/inward-open conformation (15) and allowed definition of the extracellular gate, a model in the "alternate" conformation was required to define the structural components of the intracellular gate. Because ENT or SLC29 family members appear to fold similarly to MFS family permeases (12-15), we employed homology modeling of LdNT1.1 to the only member of the MFS whose structure has been solved in an inwardclosed conformation, the E. coli FucP transporter (Fig.…”

“…The ab initio computational studies on the LdNT1.1 adenosine/pyrimidine nucleoside transporter of L. donovani (15) captured this protein in a conformation that is "closed to the outside, open to the inside," similar in the disposition of TMs and conformation to that observed in the crystal structures of the Escherichia coli lactose permease (LacY) (16) and glycerol 3-phosphate transporter (GlpT) (17), both members of the MFS family. The ab initio model predicted that most of the LdNT1.1 mutants that we previously found to greatly impair transport activity or alter substrate specificity (15,18) line the central permeation pore, as might be expected for mutations that induce strong transport phenotypes. In addition, analogous to the experimentally determined structures for the bacterial permeases LacY and GlpT, the LdNT1.1 ab initio model predicted that TM helices 1, 2, and 7 cluster together at the extracellular surface of the transporter to close off the pore or permeation pathway and form an "extracellular gate" (15,19).…”

“…These observations allowed the construction, by computational approaches, of putative tertiary structures of several protozoan ENTs, including Plasmodium falciparum PfENT1 (12), Trypanosoma brucei TbNBT1 (13), and Leishmania donovani LdNT2 (14). and LdNT1.1 (15). The ab initio computational studies on the LdNT1.1 adenosine/pyrimidine nucleoside transporter of L. donovani (15) captured this protein in a conformation that is "closed to the outside, open to the inside," similar in the disposition of TMs and conformation to that observed in the crystal structures of the Escherichia coli lactose permease (LacY) (16) and glycerol 3-phosphate transporter (GlpT) (17), both members of the MFS family.…”

“…Given the success in defining the extracellular gate of LdNT1.1 (15,19), the present work aimed to elucidate the structural components that constitute the intracellular gate located on the opposite or cytoplasmic face of the permease. The rationale for this work was that together these studies identify two critical structural components of this ENT family permease, the two gates that define the outward-closed/inward-open and inward-closed/outward-open conformations.…”