Purine substrate recognition by the nucleobase-ascorbate transporter signature motif in the YgfO xanthine permease: ASN-325 binds and ALA-323 senses substrate
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Ημερομηνία
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peer-reviewed
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J Biol Chem
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Περιγραφή
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.
Περιγραφή
Λέξεις-κλειδιά
Alanine/*chemistry, Amino Acid Motifs, Amino Acid Sequence, Ascorbic Acid/*chemistry, Asparagine/*chemistry, Escherichia coli/metabolism, Escherichia coli Proteins/chemistry/*physiology, Ethylmaleimide/chemistry, Inhibitory Concentration 50, Kinetics, Mesylates/chemistry, Molecular Sequence Data, Mutation, Nucleic Acids/chemistry, Nucleobase Transport Proteins/chemistry/*physiology, Protein Structure, Secondary, Purines/chemistry, Sequence Homology, Amino Acid
Θεματική κατηγορία
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http://www.ncbi.nlm.nih.gov/pubmed/20406814
http://www.jbc.org/content/285/25/19422.full.pdf
http://www.jbc.org/content/285/25/19422.full.pdf
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en
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Γενική Περιγραφή / Σχόλια
Ίδρυμα και Σχολή/Τμήμα του υποβάλλοντος
Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής