Role of Intramembrane Polar Residues in the YgfO Xanthine Permease

Abstract

Using the YgfO xanthine permease of Escherichia coli as a bacterial model for the study of the evolutionarily ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family, we performed a systematic Cys-scanning and site-directed mutagenesis of 14 putatively charged (Asp, Glu, His, Lys, or Arg) and 7 highly polar (Gln or Asn) residues that are predicted to lie in transmembrane helices (TMs). Of 21 single-Cys mutants engineered in the background of a functional YgfO devoid of Cys residues (C-less), only four are inactive or have marginal activity (H31C, N93C, E272C, D304C). The 4 residues are conserved throughout the family in TM1 (His-31), TM3 (Asn-93/Ser/Thr), TM8 (Glu-272), and putative TM9a (Asp-304/Asn/Glu). Extensive site-directed mutagenesis in wild-type background showed that H31N and H31Q have high activity and affinity for xanthine but H31Q recognizes novel purine bases and analogues, whereas H31C and H31L have impaired affinity for xanthine and analogues, and H31K or H31R impairs expression in the membrane. N93S and N93A are highly active but more promiscuous for recognition of analogues at the imidazole moiety of substrate, N93D has low activity, N93T has low affinity for xanthine or analogues, and N93Q or N93C is inactive. All mutants replacing Glu-272 or Asp-304, including E272D, E272Q, D304E, and D304N, are inactive, although expressed to high levels in the membrane. Finally, one of the 17 assayable single-Cys mutants, Q258C, was sensitive to inactivation by N-ethylmaleimide. The findings suggest that polar residues important for the function of YgfO cluster in TMs 1, 3, 8 and 9a.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 cellular uptake of uracil, xanthine, or uric acid (microbial and plant genomes) or vitamin C (mammalian genomes) (1, 2). Despite their importance 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 1000 sequence entries are known, but few have been functionally characterized to date. The best studied eukaryotic member is UapA, a high affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (3-7). Studies with chimeric transporter constructs (3), site-directed mutagenesis, second-site suppressors, and kinetic inhibition analysis of ligand specificity have shown that a conserved NAT/NCS2 motif region between putative transmembrane helices 8 and 9 of UapA includes determinants of substrate recognition and selectivity, with at least one residue (Gln-408) implicated in binding with the imidazole moiety of purine (4), whereas a conserved QH motif at the middle of TM1 is important for activity and/or correct targeting to the plasma membrane (5), and an aromatic residue at the middle of TM12 (Phe-528) may act as a purine subst...

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

“…Active Xanthine Transport-Using a functional YgfO permease devoid of Cys residues (C-less), each amino acid residue in sequences 259 FLVVGTIYLLSVLEAVGDITATAMVSRRPI-QGEEYQSRLKGGVLADGLVSVIASAV 314 and 342 TIAVML-VILGLFP 354 including putative amphipathic helices TM8, TM9a, and TM9b (underlined) was replaced individually with Cys, except Glu-272 and Asp-304, which had been studied by us previously (10). After verification of the sequence, each single Cys mutant was transformed into E. coli T184 and assayed for its ability to catalyze active xanthine transport.…”

“…carboxylate on substrate binding. Similarly, the functionally irreplaceable Glu-272, located one ␣-helical turn upstream of Asp-276 in putative TM8, does not appear to be essential for high-affinity binding per se but, rather, for the conformational changes following binding or coupling purine with proton translocation (10). In addition, single Cys G275C, which falls on the same ␣-helical face as Glu-272 and Asp-276 (Fig.…”

“…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.…”