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

Abstract

SummaryThe 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.

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli

“…Protein preparation using OPLS2005 force field (Banks et al ., ) and molecular docking was performed with the Schrödinger Suite 2016, as described in Karena et al . (). Substrates were docked on the most dominant cluster representative receptor structures, using the induced fit docking (IFD) protocol (Schrödinger Release 2016‐4: Schrödinger Suite 2016‐4 Induced Fit Docking protocol; Glide, Schrödinger, LLC, New York, NY, 2016; Prime, Schrödinger, LLC, New York, NY, 2016.…”

“…NAT/NCS2 transporters are present in all domains of life and include ion-gradient driven transporters of key metabolites or anti-metabolite analogs with diverse substrate preferences, ranging from purine or pyrimidine permeases in various organisms to Na 1 -dependent vitamin C transporters in human and other mammals (Gournas et al, 2008;Yamamoto et al, 2010;Frillingos, 2012;B€ urzle et al, 2013;Girke et al, 2014). Research on this family has commanded attention recently with the elucidation of high-resolution structures for two homologs, the uracil permease UraA of E. coli (Lu et al, 2011) and the xanthine/uric acid permease UapA of Aspergillus nidulans (Alguel et al, 2016), coupled with functional insight from extensive mutagenesis studies on UapA (Diallinas, 2016) and the xanthine permease XanQ of E. coli (Frillingos, 2012;Karena et al, 2015).…”

“…Few permeases of the NAT/NCS2 family have been analyzed rigorously for their substrate specificity profiles and their key specificity determinants. The available evidence comes mostly from the extensive mutagenesis work on UapA and XanQ, and few targeted studies on other purine-transporting homologs in A. nidulans and E. coli (Gournas et al, 2008;Frillingos, 2012;Papakostas and Frillingos, 2012;Papakostas et al, 2013;Krypotou et al, 2014;Karena et al, 2015;Diallinas, 2016). These studies emphasize that the roles of some key binding-site residues are conserved despite a significant variation in specificities.…”

“…These studies emphasize that the roles of some key binding‐site residues are conserved despite a significant variation in specificities. In addition, important residues affecting the specificity are found at the binding site periphery and at distal sites which may contribute to specificity by controlling the access of different substrates to the binding site (Karena et al ., ; Diallinas, ). Interestingly, such insight is not available on UraA or any of the putative pyrimidine‐transporting homologs in the UraA cluster.…”

NAT/NCS2-hound: A Webserver for the detection and evolutionary classification of prokaryotic and eukaryotic nucleobase–cation symporters of the NAT/NCS2 family

“…The first one, COG2233 or NAT, contains bacterial and fungal permeases for purines (xanthine, uric acid), bacterial permeases for pyrimidines (uracil, thymine), plantal and mammalian broad-specificity uracil/purine permeases (not present in human), and the mammalian L-ascorbate transporters SVCT1 and SVCT2. Insight on the transport mechanism of this subfamily has been provided by high-resolution crystal structures for two members, the uracil permease UraA of E. coli [16,18] and the xanthine/uric acid permease UapA of Aspergillus nidulans [17], coupled with extensive mutagenesis studies on UapA [21], the xanthine permease XanQ of E. coli [1,22] and few other homologs [23,24]. The other subfamily, COG2252 or AzgA-like [25], contains bacterial, fungal and plantal permeases for salvageable purines (adenine, guanine, hypoxanthine) which are less well studied with respect to structure-function relationships [7,26].…”