Dynamic Elements at Both Cytoplasmically and Extracellularly Facing Sides of the UapA Transporter Selectively Control the Accessibility of Substrates to Their Translocation Pathway

Abstract

In the UapA uric acid-xanthine permease of Aspergillus nidulans, subtle interactions between key residues of the putative substrate binding pocket, located in the TMS8-TMS9 loop (where TMS is transmembrane segment), and a specificity filter, implicating residues in TMS12 and the TMS1-TMS2 loop, are critical for function and specificity. By using a strain lacking all transporters involved in adenine uptake (DeltaazgA DeltafcyB DeltauapC) and carrying a mutation that partially inactivates the UapA specificity filter (F528S), we obtained 28 mutants capable of UapA-mediated growth on adenine. Seventy-two percent of mutants concern replacements of a single residue, R481, in the putative cytoplasmic loop TMS10-TMS11. Five missense mutations are located in TMS9, in TMS10 or in loops TMS1-TMS2 and TMS8-TMS9. Mutations in the latter loops concern residues previously shown to enlarge UapA specificity (Q113L) or to be part of a motif involved in substrate binding (F406Y). In all mutants, the ability of UapA to transport its physiological substrates remains intact, whereas the increased capacity for transport of adenine and other purines seems to be due to the elimination of elements that hinder the translocation of non-physiological substrates through UapA, rather than to an increase in relevant binding affinities. The additive effects of most novel mutations with F528S and allele-specific interactions of mutation R481G (TMS10-TMS11 loop) with Q113L (TMS1-TMS2 loop) or T526M (TMS12) establish specific interdomain synergy as a critical determinant for substrate selection. Our results strongly suggest that distinct domains at both sides of UapA act as selective dynamic gates controlling substrate access to their translocation pathway.

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

“…It is interesting to note that Thr-280 and, especially, Ala-279 display a high degree of conservation among members of the NAT/NCS2 family (5,10) and that Ala-363, the residue corresponding to Ala-279 in the fungal homolog UapA (5), has been suggested as critical for function in Aspergillus nidulans, based on the loss-of-function phenotype of mutant A363D (18). Although a more comprehensive mutagenesis study of Ala-363 is missing (5,6), genetic analysis has shown that presence of a Gly in place of Ala-363 is compatible with full UapA activity (18), which is reminiscent of the wild-type expression and activity seen with mutant A279G in YgfO (Fig. 6).…”

“…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).…”

The Role of Transmembrane Segment TM3 in the Xanthine Permease XanQ of Escherichia coli

“…Homology modeling shows that most of the residues identified as important in both XanQ (7)(8)(9) and UapA (11)(12)(13)(14) fall in TM1, TM3, TM8, and the NAT-signature motif (TM10) and are either in the putative binding site or at the periphery forming a shelter around substrate in the core domain of the protein (supplemental Fig. S1).…”

“…These residues are probably crucial in defining the molecular basis of NAT substrate recognition and selectivity. In addition, more dynamic structural elements located in the gate domain (1) may contribute to affinity and specificity by controlling access of substrates to the binding site and implementing the appropriate conformational changes; the current genetic and biochemical evidence on UapA (12)(13)(14) and XanQ (6) suggests that residues involved in such gating mechanisms are found in the last transmembrane segment (TM14) (6,12,13), in cytoplasmic loop TM12-TM13 (13) and in sequence regions flanking TM1 (13,14).…”

Substrate Selectivity of YgfU, a Uric Acid Transporter from Escherichia coli

“…With respect to function, only 15 of more than 2,000 predicted members have been characterized in detail; these are specific for the cellular uptake of uracil, xanthine, or uric acid (microbial, plant, and nonprimate mammalian genomes) or vitamin C (mammalian genomes) (1)(2)(3). Two of them, the xanthine permease XanQ of Escherichia coli (4 -10) and the uric acid/xanthine permease UapA of Aspergillus nidulans (11)(12)(13)(14)(15), have been studied extensively with Cysscanning mutagenesis and reverse and forward genetics, respectively. These studies have shown striking similarities between key NAT determinants of the two transporters, reinforcing the idea that few residues at conserved motifs of the family may be invariably critical for function or underlie specificity differences (for a summary of current knowledge, see supplemental Fig.…”