Purine Substrate Recognition by the Nucleobase-Ascorbate Transporter Signature Motif in the YgfO Xanthine Permease

Abstract

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved 11-amino acid sequence of the ubiquitous NAT/ NCS2 family, essential for function and selectivity of both a bacterial (YgfO) and a fungal (UapA) purine-transporting homolog. We examined the role of NAT motif in more detail, using Cys-scanning and site-directed alkylation analysis of the YgfO xanthine permease of Escherichia coli. Analysis of single-Cys mutants in the sequence 315-339 for sensitivity to inactivation by 2-sulfonatoethyl methanethiosulfonate (MTSES ؊ ) and N-ethylmaleimide (NEM) showed a similar pattern: highly sensitive mutants clustering at the motif sequence (323-329) and a short ␣-helical face downstream (332, 333, 336). In the presence of substrate, N325C is protected from alkylation with either MTSES ؊ or NEM, whereas sensitivity of A323C to inactivation by NEM is enhanced, shifting IC 50 from 34 to 14 M. Alkylation or sensitivity of the other mutants is unaffected by substrate; the lack of an effect on Q324C is attributed to gross inability of this mutant for high affinity binding. Site-directed mutants G333R and S336N at the ␣-helical face downstream the motif display specific changes in ligand recognition relative to wild type; G333R allows binding of 7-methyl and 8-methylxanthine, whereas S336N disrupts affinity for 6-thioxanthine. Finally, all assayable motif-mutants are highly accessible to MTSES ؊ from the periplasmic side. The data suggest that the NAT motif region lines the solvent-and substrate-accessible inner cavity, Asn-325 is at the binding site, Ala-323 responds to binding with a specific conformational shift, and Gly-333 and Ser-336 form part of the purine permeation pathway.

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

“…More than 1,000 sequence entries are known but few are functionally characterized to date. Structure-function relationships have been studied extensively in two members, the eukaryotic UapA, a high-affinity uric acid/xanthine:H ϩ symporter from the ascomycote Aspergillus nidulans (3)(4)(5)(6) and the prokaryotic YgfO, a specific, high-affinity xanthine:H ϩ symporter from Escherichia coli (7)(8)(9)(10)(11). Mutagenesis data from both lines of study have shown that key NAT determinants are strikingly similar between the two transporters, and that few residues conserved throughout the family may be invariably critical for function (10).…”

“…1). 3 Of them, Asn-325 and Gln-324 belong to the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, and site-directed alkylation analysis suggests that they participate directly in the substrate binding site (11). The other two irreplaceable residues of YgfO are in the neighboring helices TM8 (Glu-272) and TM9a (Asp-304) and appear to be implicated with the conformational changes following binding and/or coupling purine with proton translocation, but not with substrate binding per se (10).…”

“…The other two irreplaceable residues of YgfO are in the neighboring helices TM8 (Glu-272) and TM9a (Asp-304) and appear to be implicated with the conformational changes following binding and/or coupling purine with proton translocation, but not with substrate binding per se (10). Apart from the irreplaceable ones, a number of other important residues have been found, including residues at the middle of helices TM1 (His-31) and TM3 (Asn-93), which contribute to determining the proper affinity and selectivity of the purine binding site (10), residues at the middle of TM12 (Asn-430, Ile-432), which are close to the binding site and/or optimize binding indirectly (9), and residues within or adjacent to the NAT motif which are important conformationally (Ala-323) (11) or involved in defining the optimal specificity profile (Thr-332, Gly-333, Ser-336) (8,11). Overall, residues delineated as crucial for the mechanism of substrate recognition and selectivity cluster primarily at the NAT motif region (Fig.…”

“…Recently, Cys-scanning and substituted-Cys accessibility analysis (11) has shown that the NAT motif of YgfO permease may form a re-entrant loop between helices TM9a and TM9b that faces the central hydrophilic cavity and is accessible to substrate and solvent from the periplasmic side (Fig. 1).…”

“…) and [8][9][10][11][12][13][14] C]uric acid (51.5 mCi mmol Ϫ1 ) were purchased from Moravek Biochemicals. Non-radioactive nucleobases and analogues were from Sigma.…”