Cysteine-scanning Analysis of Putative Helix XII in the YgfO Xanthine Permease

Abstract

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...

Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

“…Segments of the YgfO sequence are shown as helical wheel plots of residues 259 -276 (TM8), 301-318 (TM9a), 329 -346 (TM9b), and 419 -436 (TM12). The four irreplaceable residues (Glu-272, Asp-304, Gln-324, Asn-325) (red), Asp-276, which contains an essential carboxyl group (red, underlined) and Asn-430, which is vicinal to the binding site (9) are enlarged and bolded and shown as targets. Sites of NEM-sensitive single Cys mutants are shown with a dark background, and highly sensitive sites (IC 50 Յ 30 M) are shown with bolder peripheries.…”

“…The other two irreplaceable residues of YgfO are in the neighboring helices TM8 (Glu-272) and TM9a (Asp-304) and appear to be implicated with the conformational changes following binding and/or coupling purine with proton translocation, but not with substrate binding per se (10). Apart from the irreplaceable ones, a number of other important residues have been found, including residues at the middle of helices TM1 (His-31) and TM3 (Asn-93), which contribute to determining the proper affinity and selectivity of the purine binding site (10), residues at the middle of TM12 (Asn-430, Ile-432), which are close to the binding site and/or optimize binding indirectly (9), and residues within or adjacent to the NAT motif which are important conformationally (Ala-323) (11) or involved in defining the optimal specificity profile (Thr-332, Gly-333, Ser-336) (8,11). Overall, residues delineated as crucial for the mechanism of substrate recognition and selectivity cluster primarily at the NAT motif region (Fig.…”

“…More than 1,000 sequence entries are known but few are functionally characterized to date. Structure-function relationships have been studied extensively in two members, the eukaryotic UapA, a high-affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (3)(4)(5)(6) and the prokaryotic YgfO, a specific, high-affinity xanthine:H ϩ symporter from Escherichia coli (7)(8)(9)(10)(11). Mutagenesis data from both lines of study have shown that key NAT determinants are strikingly similar between the two transporters, and that few residues conserved throughout the family may be invariably critical for function (10).…”

“…) and [8][9][10][11][12][13][14] C]uric acid (51.5 mCi mmol Ϫ1 ) were purchased from Moravek Biochemicals. Non-radioactive nucleobases and analogues were from Sigma.…”

Role of Intramembrane Polar Residues in the YgfO Xanthine Permease

“…We launched a systematic series of Cys-scanning and site-directed mutagenesis studies of YgfO to elucidate structure-function relationships in a bacterial NAT (9,10). In the course of these studies, we showed that the NAT motif sequence region of YgfO includes the essential determinants Gln-324, irreplaceable for high affinity binding and uptake; Asn-325, irreplaceable for active transport; and an ␣-helical stripe of residues (Thr-332, Gly-333, Ser-336, Val-339), highly sensitive to site-directed alkylation and important for ligand selectivity 3 (9).…”