Role of transmembrane domain 4 in ligand permeation by Crithidia fasciculata equilibrative nucleoside transporter 2 (CfNT2)

Abstract

Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.

FIGURE 1.

Predicted topology of residues that influence ENT ligand specificity. The generic topology of an ENT family member is depicted, and N (NH₂) and C (COOH) termini as well as intracellular (in) and extracellular (out) sides are indicated. Transmembrane helices predicted to lie on the exterior of the protein structure are shaded. Residues identified in this work are indicated in black (Lys¹⁵⁵) and white (Gly⁸⁶ and Asp¹⁵⁹) circles. The predicted locations of residues described in the literature as influencing ligand specificity are indicated by gray circles (TM1, hENT2-I33 (51, 52) and hENT1-W29 (53); TM2, hENT1-L92 (54); TM4, LdNT1.1-K153 (15); TM5, LdNT1.1-G183 (55) and rENT1/rENT2 chimeras (56)).

FIGURE 2.

Substitutions at Lys¹⁵⁵ of CfNT2 confer adenosine transport activity. Purine auxotrophic yeast cells (Δade2) expressing the indicated ORFs under the control of the yeast CUP1 promoter were grown on SD ura⁻ ade⁻ medium supplemented with 100 μm CuSO₄ and inosine (A), adenosine (B), or adenosine and guanosine (C) as purine source(s). Plates were photographed after 3 days of growth at 30 °C.

FIGURE 3.

Cell surface labeling of cfnt2-K155 mutants expressed in L. donovani. 4 × 10⁸ Δldnt1/Δldnt2 cells transfected with pALTneo carrying the indicated allele or with empty plasmid were treated with biotin as described under “Experimental Procedures.” Biotinylated proteins were bound to streptavidin beads and separated on a 10% SDS-polyacrylamide gel. Western blots were performed with anti-HA and anti-MIT antibodies, and proteins were visualized with chemiluminescence. Locations of size markers in kDa are indicated on the right, and the positions of HA-cfnt2 and the MIT protein (loading control) are indicated on the left. Relative intensities of the HA-cfnt2 bands were estimated from densitometry of scanned films.

FIGURE 4.

Biochemical and kinetic characterization of cfnt2-K155N. Δldnt1/Δldnt2 pALTneo-HA-cfnt2-K155N cells were incubated with varying concentrations of [³H]inosine (A) or [³H]adenosine (B), and transport was measured over linear 2-min time courses by the oil-stop method. Michaelis-Menten kinetics of representative experiments (n = 3) are depicted. C, the same cell line was incubated with 5 μm [³H]inosine in the presence of 500 μm unlabeled purine or pyrimidine, and triplicate transport measurements were made at 30 s. A representative experiment is shown (n = 2).

FIGURE 5.

Predicted topology of CfNT2 TM4. A, alignment of protozoan and human ENT proteins around predicted TM4. The filled arrowheads indicate residues in CfNT2 shown by SCAM to be water-accessible; the open arrowhead indicates Lys¹⁵⁵, which influences ligand specificity. The line below the alignment indicates a published prediction for the residues included in TM4 in human transporters hENT1 and hENT2 (11), which is consistent with these results. The line above the alignment indicates a previous prediction for the topology of CfNT2 TM4 (17). GenBank^TM accession numbers are as follows: CfNT2 (AAG22611), LdNT2 (AAF74264), LdNT1.1 (AAC32597), TbNT2 (AAF04490), TbAT1 (AAB93848), hENT1 (AAC51103), hENT2 (NP_001523). The GeneDB accession number for LmajNT3 is LmjF13.1210. B, helical wheel diagram of residues 151–170 from CfNT2. Positions that were observably modified with MTSEA and/or MTSES in the SCAM analysis are highlighted in gray. Those whose modification could be partially blocked by the presence of ligand are highlighted in black.

FIGURE 6.

Effect of MTSEA treatment of cfnt2ΔCys-K155C on inosine uptake capability. A, uptake of over 3 min was measured in triplicate by Δldnt1/Δldnt2 pALTneo-HA-cfnt2ΔCys-K155C cells under a variety of conditions. All cells were first treated or mock-treated with 2.5 mm MTSEA or 10 mm MTSES for 10 min in the absence or presence (+ ligand) of 1 mm inosine. Following three washes of the cells to remove unreacted reagent and excess unlabeled ligand, uptake of 5 μm [³H]inosine was measured. No treatment, cells not treated with thiol-modifying reagents (relative uptake set to 100%); competitive inhibitor, mock-treated cells whose uptake of 5 μm [³H]inosine was challenged with 5 mm unlabeled inosine. B, structures of MTSEA as well as MTSEA-modified cysteine (Cys adduct) and lysine side chains.

FIGURE 7.

Structure-activity analysis. Δldnt1/Δldnt2 cells expressing CfNT2 (A) or cfnt2-K155N (B) were incubated with 5 μm [³H]inosine in the presence of a 500 μm concentration of the indicated purine analog in parallel with uninhibited cells (control) in duplicate at a time point within the linear range of uptake (7 s for CfNT2 and 60 s for cfnt2-K155N). Relative uptake compared with uninhibited cells is shown. Data represent at least two independent determinations with each inhibitor. C, the structure of inosine with purine nucleoside numbering scheme indicated. The 2-amino group of guanosine is indicated in parentheses.