Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate.
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 alpha-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 microM. 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 alpha-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.
Georgopoulou
Ekaterini
E
Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece.
Mermelekas
George
G
Karena
Ekaterini
E
Frillingos
Stathis
S
D000409:Alanine / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000737:chemistry*; D001216:Asparagine / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D005033:Ethylmaleimide / Q000737:chemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008698:Mesylates / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009696:Nucleic Acids / Q000737:chemistry; D033702:Nucleobase Transport Proteins / Q000737:chemistry / Q000502:physiology*; D017433:Protein Structure, Secondary; D011687:Purines / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D029968:Escherichia coli Proteins; D008698:Mesylates; D009696:Nucleic Acids; D033702:Nucleobase Transport Proteins; D011687:Purines; C529797:YgfO protein, E coli; C109153:(2-sulfonatoethyl)methanethiosulfonate; D001216:Asparagine; D005033:Ethylmaleimide; D000409:Alanine; D001205:Ascorbic Acid
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