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The nucleobase-ascorbate transporter (NAT) signature motif is a conserved 11-amino acid sequence of the ubiquitous NAT/ NCS2 family, essential for function and selectivity of both a bacterial (YgfO) and a fungal (UapA) purine-transporting homolog. We examined the role of NAT motif in more detail, using Cys-scanning and site-directed alkylation analysis of the YgfO xanthine permease of Escherichia coli. Analysis of single-Cys mutants in the sequence 315-339 for sensitivity to inactivation by 2-sulfonatoethyl methanethiosulfonate (MTSES ؊ ) and N-ethylmaleimide (NEM) showed a similar pattern: highly sensitive mutants clustering at the motif sequence (323-329) and a short ␣-helical face downstream (332, 333, 336). In the presence of substrate, N325C is protected from alkylation with either MTSES ؊ or NEM, whereas sensitivity of A323C to inactivation by NEM is enhanced, shifting IC 50 from 34 to 14 M. Alkylation or sensitivity of the other mutants is unaffected by substrate; the lack of an effect on Q324C is attributed to gross inability of this mutant for high affinity binding. Site-directed mutants G333R and S336N at the ␣-helical face downstream the motif display specific changes in ligand recognition relative to wild type; G333R allows binding of 7-methyl and 8-methylxanthine, whereas S336N disrupts affinity for 6-thioxanthine. Finally, all assayable motif-mutants are highly accessible to MTSES ؊ from the periplasmic side. The data suggest that the NAT motif region lines the solvent-and substrate-accessible inner cavity, Asn-325 is at the binding site, Ala-323 responds to binding with a specific conformational shift, and Gly-333 and Ser-336 form part of the purine permeation pathway.

Transmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cysscanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 419 ILPASIYVLVENPICAGGLTAILLNIILPGGY 450 (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80 -130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15-40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC 50 of 10 M, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site.The nucleobase-ascorbate transporter (NAT) 2 or nucleobase-cation symporter-2 (NCS2) family is an evolutionarily ubiquitous family of purine, pyrimidine, and L-ascorbate transporters, with members specific for the cellular uptake of uracil, xanthine, or uric acid (microbial and plant genomes) or vitamin C (mammalian genomes) (1-3). Despite their importance as molecular gateways for the recognition and uptake of several frontline purine-related drugs, NAT/NCS2 members have not been studied systematically at the molecular level, and high resolution structures or mechanistic models are missing. More than 500 sequence entries are known, but few are functionally characterized in detail.The first NAT to be sequenced and best studied eukaryotic member to date is UapA, a high affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (4). Studies with chimeric transporter constructs, site-directed mutagenesis, second-site suppressors, and kinetic inhibition analysis of ligand specificity (5-8) have shown that a conserved NAT/NCS2 motif region between putative transmembrane helices VIII and IX of UapA includes determinants of substrate recognition and selectivity (8), with at least one residue (Gln-408) implicated in purine binding with the imidazole moiety of purines, wh...

Bacterial and fungal members of the ubiquitous nucleobaseascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cysscanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences 259 FLVVG-TIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLAD-GLVSVIASAV 314 and 342 TIAVMLVILGLFP 354 including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35-140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10 -20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (K i 79 M) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC 50 15 M) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXG-DXXAT) and in TM9a (GXXXDG).

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