Identification of New Specificity Determinants in Bacterial Purine Nucleobase Transporters based on an Ancestral Sequence Reconstruction Approach.

The relation of sequence with specificity in membrane transporters is challenging to explore. Most relevant studies until now rely on comparisons of present-day homologs. In this work, we study a set of closely related transporters by employing an evolutionary, ancestral-reconstruction approach and reveal unexpected new specificity determinants. We analyze a monophyletic group represented by the xanthine-specific XanQ of Escherichia coli in the Nucleobase-Ascorbate Transporter/Nucleobase-Cation Symporter-2 (NAT/NCS2) family. We reconstructed AncXanQ, the putative common ancestor of this clade, expressed it in E. coli K-12, and found that, in contrast to XanQ, it encodes a high-affinity permease for both xanthine and guanine, which also recognizes adenine, hypoxanthine, and a range of analogs. AncXanQ conserves all binding-site residues of XanQ and differs substantially in only five intramembrane residues outside the binding site. We subjected both homologs to rationally designed mutagenesis and present evidence that these five residues are linked with the specificity change. In particular, we reveal Ser377 of XanQ (Gly in AncXanQ) as a major determinant. Replacement of this Ser with Gly enlarges the specificity of XanQ towards an AncXanQ-phenotype. The ortholog from Neisseria meningitidis retaining Gly at this position is also a xanthine/guanine transporter with extended substrate profile like AncXanQ. Molecular Dynamics shows that the S377G replacement tilts transmembrane helix 12 resulting in rearrangement of Phe376 relative to Phe94 in the XanQ binding pocket. This effect may rationalize the enlarged specificity. On the other hand, the specificity effect of S377G can be masked by G27S or other mutations through epistatic interactions.

Analysis of conserved NCS2 motifs in the Escherichia coli xanthine permease XanQ.

The xanthine permease XanQ of Escherichia coli is a paradigm for transporters of the evolutionarily broad family nucleobase-cation symporter-2 (NCS2) that 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.