Bacterial and fungal members of the ubiquitous nucleobase-ascorbate 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 Cys-scanning 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)FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV(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 μm) 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 μm) 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 (EXXGDXXAT) and in TM9a (GXXXDG).
"In our previous Cys-scanning studies of XanQ, we have focused on polar residues predicted to fall in TMs (Karena and Frillingos, 2009) and on large sequence segments flanking the important polar residues in TM3 (Karena and Frillingos, 2011), TM8-TM11 (Georgopoulou et al., 2010; Mermelekas et al., 2010) and TM14 (Papakostas et al., 2008). Those studies have delineated several key residues at or around the binding site of the homology modeled structure, including the irreplaceable and highly "
[Show abstract][Hide abstract] ABSTRACT: The xanthine permease XanQ of Escherichia coli is a paradigm for transporters of the evolutionarily broad family nucleobase-cation symporter-2 (NCS2) which transport key metabolites or anti-metabolite analogs. Most functionally known members are xanthine/uric acid transporters related to XanQ and belong to a distinct phylogenetic cluster of the family. Here, we present a comprehensive mutagenesis of XanQ based on the identification and Cys-scanning analysis of conserved sequence motifs in this cluster. Results are interpreted in relation to homology modeling on the structurally known template of UraA and previous data on critical binding-site residues in transmembrane segments (TMs) 3, 8, and 10. The current analysis, of motifs distant to the binding site, revealed a set of functionally important residues in TMs 2, 5, 12 and 13, including seven irreplaceable ones, of which six are Gly residues in the gate domain (159, 369, 370, 383, 409) and in TM2 (Gly-71), and one is polar (Gln-75). Gln-75 (TM2) is probably crucial in a network of hydrogen-bonding interactions in the middle of the core domain involving another essential residue, Asp-304 (TM9). Although the two residues are irreplaceable individually, combinatorial replacement of Gln-75 with Asn and of Asp-304 with Glu rescues significant transport activity.
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"Interestingly, in primates, NATs are solely specific for L-ascorbic acid (vitamin C) transport. Structure– function relationships of two NAT proteins, UapA from the ascomycete Aspergillus nidulans (Diallinas et al., 1998; Meintanis et al., 2000; Koukaki et al., 2005; Vlanti et al., 2006; Pantazopoulou and Diallinas, 2007; Papageorgiou et al., 2008; Kosti et al., 2010; Amillis et al., 2011) and XanQ from Escherichia coli (Georgopoulou et al., 2010; Mermelekas et al., 2010; Frillingos, 2012) have been studied in great detail. These studies have led to the identification of amino acids critical for substrate recognition and transport. "
[Show abstract][Hide abstract] ABSTRACT: The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterised NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterised mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high capacity, H+/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.
"The structural model of UapA and subsequent docking and MD approaches showed that a major xanthine binding site is formed by four specific residues in TMS3 (Phe155), TMS8 (Glu356) and TMS10 (Ala407 and Gln 408). The same residues were shown to bind xanthine in the homologous E. coli XanQ permease (Karatza et al., 2006; Karena and Frillingos, 2009, 2011; Georgopoulou et al., 2010; Mermelekas et al., 2010). "
[Show abstract][Hide abstract] ABSTRACT: Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action.
Frontiers in Pharmacology 09/2014; 5. DOI:10.3389/fphar.2014.00207 · 3.80 Impact Factor
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