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

Abstract

Background:The xanthine permease XanQ is a prototype of nucleobase:proton symporters of the ubiquitous family NCS2. Results: Site-directed replacements of Asn-93 and Phe-94 in the third transmembrane segment of XanQ distort the specificity for substrate. Conclusion: Asn-93 interacts with xanthine-coordinating residues in the binding pocket. Significance: Dissecting specificity determinants in family NCS2 is crucial for understanding evolution of purine uptake.

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

“…As a result, homology modeling has been used as a reasonable alternative in cases where no x-ray structure is available (23,48,59,61,(67)(68)(69)(70)(71)(72)(73). Using this approach, combined with induced fit docking and a detailed characterization of the specificity profile, we identified 9 residues that are important for PrnB function, Gly 56 Table 5).…”

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

“…Thus, the side chain of Thr-100 is linked with the transporter ability to differentiate between uric acid (8-oxyxanthine) and xanthine. A similar role has been proposed for the corresponding amino acid in permease XanQ (Asn-93) because replacement of Asn-93 with Ala or Ser converts the xanthine-selective XanQ to dual-selectivity variants (7,10); in the case of XanQ, however, the dual-selectivity variants transport the non-wild-type substrate (uric acid) with very low capacity (10). The selectivity-related role of Thr-100/Asn-93 appears to be unique because no other single-replacement mutant of either XanQ (6 -10) or YgfU (this study) has been shown to convert the native transporter to a dual-selectivity one.…”

“…2). Most importantly, as well, critical roles have been described for the corresponding residues of other known NAT homologs (1,(7)(8)(9)(10)(11)(12)(13)43), as addressed in more detail below.…”

“…Namely, the presence of an uncharged hydrogen-bonding residue at this position is essential for high-affinity binding and transport in XanQ (7) and in the hSVCTs (43,44), whereas in UapA, it has been suggested as crucial for proper folding and targeting to the plasma membrane (15). The recently elucidated crystal structure of the different-specificity homolog UraA (1) as well as homology models built on this structure (10,15) show that the conserved His of TM1 hydrogen-bonds to the conserved Asn of the NAT signature motif at the binding site periphery, substantiating further the previous findings. Based on the mutagenesis data of Fig.…”

“…S1). Based on homology modeling, most of these residues are found at the vicinity or at the periphery of the binding site in transmembrane segments TM1, TM3, TM8, and TM10 (1,10,15).…”