Cysteine-scanning analysis of helices TM8, TM9a, and TM9b and intervening loops in the YgfO xanthine permease: a carboxyl group is essential at ASP-276.
ABSTRACT 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).
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ABSTRACT: The nucleobase-ascorbate transporter or nucleobase-cation symporter-2 (NAT/NCS2) family is one of the five known families of transporters that use nucleobases as their principal substrates and the only one that is evolutionarily conserved and widespread in all major taxa of organisms. The family is a typical paradigm of a group of related transporters for which conservation in sequence and overall structure correlates with high functional variations between homologs. Strikingly, the human homologs fail to recognize nucleobases or related cytotoxic compounds. This fact allows important biomedical perspectives for translation of structure-function knowledge on this family to the rational design of targeted antimicrobial purine-related drugs. To date, very few homologs have been characterized experimentally in detail and only two, the xanthine permease XanQ and the uric acid/xanthine permease UapA, have been studied extensively with site-directed mutagenesis. Recently, the high-resolution structure of a related homolog, the uracil permease UraA, has been solved for the first time with crystallography. In this review, I summarize current knowledge and emphasize how the systematic Cys-scanning mutagenesis of XanQ, in conjunction with existing biochemical and genetic evidence for UapA and the x-ray structure of UraA, allow insight on the structure-function and evolutionary relationships of this important group of transporters. The review is organized in three parts referring to (I) the theory of use of Cys-scanning approaches in the study of membrane transporter families, (II) the state of the art with experimental knowledge and current research on the NAT/NCS2 family, (III) the perspectives derived from the Cys-scanning analysis of XanQ.International journal of biochemistry and molecular biology. 01/2012; 3(3):250-72.
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ABSTRACT: Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H(+) symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.PLoS ONE 07/2012; 7(7):e41939. · 3.53 Impact Factor
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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.