Cysteine-scanning Analysis of Helices TM8, TM9a, and TM9b and Intervening Loops in the YgfO Xanthine Permease

Abstract

Bacterial and fungal members of the ubiquitous nucleobaseascorbate 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 Cysscanning 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 FLVVG-TIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLAD-GLVSVIASAV 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 M) 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 M) 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 (EXXG-DXXAT) and in TM9a (GXXXDG).

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

“…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.…”

“…The role of another irreplaceable YgfU residue (Glu-270) might also be linked directly with binding because its counterpart in UraA (Glu-241) also forms two direct hydrogen bonds with substrate in the crystal structure (1). The fourth invariably irreplaceable residue (Asp-298 in YgfU) corresponds to a position of UraA, which is at the same depth in the membrane but distal from the binding site in the crystal structure and might have a distinct conformational role in the purine translocation mechanism, as suggested for permease XanQ (7,9). In any event, the fact that four conserved residues associated either directly or indirectly with binding are functionally irreplaceable in XanQ, YgfU (UacT), and UapA highlights the functional conservation of the purine binding site in NAT/NCS2 homologs of different selectivity.…”

“…The five irreplaceable YgfU residues are conserved in the xanthine-transporting homolog XanQ and the dual-specificity uric acid/xanthine transporter UapA, and four out of five are also irreplaceable for function in XanQ (8,9) and in UapA (12,13). The irreplaceable role of the counterparts of Gln-318 and Asn-319, which belong to the conserved NAT signature motif (11), has been linked with defining the function of the binding 1 mM, 37°C).…”

“…Each one of the 11 important residues of XanQ that differ in YgfU was replaced individually with the corresponding amino acid found in YgfU. Thus, we constructed XanQ mutants Q258S, V261T, A273S, T280L, S284V, A323S, N326V, and N430S and assayed them for expression, uptake activity, and substrate selectivity in parallel with mutants N93T (7), N93A (10), D276M (9), and G333R (8) that had been engineered by us in previous studies. As shown in Fig.…”

Using Cys-Scanning Analysis Data in the Study of Membrane Transport Proteins

“…These three potential properties can be used to define the set of important residues of the study prototype, as deduced from Cys-scanning analysis data (Karena & Frillingos, 2011;Papakostas & Frillingos, 2012). It is also unequivocally true that this set of residues represents positions with a higher degree of side chain conservation than the rest of the protein and, in cases of transporters that have been studied thoroughly, correspond to a small percentage of the total amino acids in the sequence, usually 10-15% (Frillingos et al, 1998;Georgopoulou et al, 2010;Mermelekas et al, 2010;Tamura et al, 2003). These features make this clearly defined set of residues suitable for use as a basis to select (i) new homologs for ab intio study and (ii) amino acid targets for site-specific mutagenesis in these homologs ( Figure 1).…”