Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate

J Biol Chem. 2010 Jun 18;285(25):19422-33. doi: 10.1074/jbc.M110.120543. Epub 2010 Apr 20.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alanine / chemistry*
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Ascorbic Acid / chemistry*
  • Asparagine / chemistry*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / physiology*
  • Ethylmaleimide / chemistry
  • Inhibitory Concentration 50
  • Kinetics
  • Mesylates / chemistry
  • Molecular Sequence Data
  • Mutation
  • Nucleic Acids / chemistry
  • Nucleobase Transport Proteins / chemistry
  • Nucleobase Transport Proteins / physiology*
  • Protein Structure, Secondary
  • Purines / chemistry
  • Sequence Homology, Amino Acid

Substances

  • Escherichia coli Proteins
  • Mesylates
  • Nucleic Acids
  • Nucleobase Transport Proteins
  • Purines
  • YgfO protein, E coli
  • (2-sulfonatoethyl)methanethiosulfonate
  • Asparagine
  • Ethylmaleimide
  • Alanine
  • Ascorbic Acid