PMID:20406814

Citation	Georgopoulou, E, Mermelekas, G, Karena, E and Frillingos, S (2010) 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. 285:19422-33
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.
Links	PubMed PMC2885222 Online version:10.1074/jbc.M110.120543
Keywords	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
edit table

Significance

You can help EcoliWiki by summarizing why this paper is useful
Demonstration that a conserved sequence motif of YgfO (XanQ) in at the transporter binding site, using a novel substituted-cysteine accessibility approach.

Useful Materials and Methods

You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Sulfhydryl alkylation assay to determine accessibility/reactivity of an engineered single-Cys in the presence or absence of substrate is performed with preincubation of RSO vesicles with reagent, at the appropriate conditions, followed by extraction of membrane proteins with DDM and isolation of His10-tagged permease through nickel-affinity chromatography, in-column denaturation and labeling with Oregon Green 488 maleimide.

Annotations

<annotationlinks/>

EcoliWiki Links

Add links to pages that link here (e.g. gene, product, method pages)

References

See Help:References for how to manage references in EcoliWiki.