title: Cysteine cross-linking defines the extracellular gate for the Leishmania donovani nucleoside transporter 1.1 (LdNT1.1).
abstract: Equilibrative nucleoside transporters are a unique family of proteins that enable uptake of nucleosides/nucleobases into a wide range of eukaryotes and internalize a myriad of drugs used in the treatment of cancer, heart disease, AIDs, and parasitic infections. In previous work we generated a structural model for such a transporter, the LdNT1.1 nucleoside permease from the parasitic protozoan Leishmania donovani, using ab initio computation. The model suggested that aromatic residues present in transmembrane helices 1, 2, and 7 interact to form an extracellular gate that closes the permeation pathway in the inward-open conformation. Mutation of residues Phe-48(TM1) and Trp-75(TM2) abrogated transport activity, consistent with such prediction. In this study cysteine mutagenesis and oxidative cross-linking were combined to analyze proximity relationships of helices 1, 2, and 7 in LdNT1.1. Disulfide bond formation between introduced paired cysteines at the interface of such helices (A61C(TM1)/F74C(TM2), A61C(TM1)/G350C(TM7), and F74C(TM2)/G350C(TM7)) was analyzed by transport measurement and gel mobility shifts upon oxidation with Cu (II)-(1,10-phenanthroline)(3). In all cases cross-linking inhibited transport. However, if LdNT1.1 ligands were included during cross-linking, inhibition of transport was reduced, suggesting that ligands moved the three gating helices apart. Moreover, all paired cysteine mutants exhibited a mobility shift upon oxidation, corroborating the formation of a disulfide bond. These data support the notion that helices 1, 2, and 7 constitute the extracellular gate of LdNT1.1, thus further validating the computational model and the previously demonstrated importance of F48(TM1) and Trp-75(TM2) in tethering together helices that are part of the gate.
authors.affiliation: Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon 97239, USA.
authors.forename: Raquel
authors.initials: R
authors.lastname: Valdés
authors.forename: Ujwal
authors.initials: U
authors.lastname: Shinde
authors.forename: Scott M
authors.initials: SM
authors.lastname: Landfear
mesh_terms: D020816:Amino Acid Motifs; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007893:Leishmania donovani / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009705:Nucleosides / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D009705:Nucleosides; D015800:Protozoan Proteins; D003545:Cysteine