Cysteine-scanning analysis of putative helix XII in the YgfO xanthine permease: ILE-432 and ASN-430 are important

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dc.contributor.authorPapakostas, K.en
dc.contributor.authorGeorgopoulou, E.en
dc.contributor.authorFrillingos, S.en
dc.date.accessioned2015-11-24T19:25:39Z
dc.date.available2015-11-24T19:25:39Z
dc.identifier.issn0021-9258-
dc.identifier.urihttps://olympias.lib.uoi.gr/jspui/handle/123456789/22644
dc.rightsDefault Licence-
dc.subjectAmino Acid Sequenceen
dc.subjectAsparagine/chemistryen
dc.subjectBinding Sitesen
dc.subjectEscherichia coli/metabolismen
dc.subjectEscherichia coli Proteins/*chemistry/geneticsen
dc.subjectEthylmaleimide/pharmacologyen
dc.subjectGene Expression Regulation, Bacterialen
dc.subjectInhibitory Concentration 50en
dc.subjectIsoleucine/chemistryen
dc.subjectKineticsen
dc.subjectMembrane Transport Proteins/*chemistry/*geneticsen
dc.subjectModels, Biologicalen
dc.subjectMolecular Sequence Dataen
dc.subjectNucleobase Transport Proteins/*chemistry/geneticsen
dc.subjectProtein Conformationen
dc.subjectProtein Structure, Secondaryen
dc.subjectSequence Homology, Amino Aciden
dc.titleCysteine-scanning analysis of putative helix XII in the YgfO xanthine permease: ILE-432 and ASN-430 are importanten
heal.abstractTransmembrane helix XII of UapA, the major fungal homolog of the nucleobase-ascorbate transporter (NAT/NCS2) family, has been proposed to contain an aromatic residue acting as a purine-selectivity filter, distinct from the binding site. To analyze the role of helix XII more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 419ILPASIYVLVENPICAGGLTAILLNIILPGGY450 (the putative helix XII is underlined) was replaced individually with Cys. Of the 32 single-Cys mutants, 25 accumulate xanthine to 80-130% of the steady state observed with C-less YgfO, six (P421C, S423C, I424C, Y425C, L427C, G436C) accumulate to low levels (15-40%), and I432C is inactive. Immunoblot analysis shows that P421C and I432C display low expression in the membrane. Extensive mutagenesis reveals that replacement of Ile-432 with equally or more bulky side chains abolishes active transport without affecting expression, whereas replacement with smaller side chains allows activity but impairs affinity for the analogues 1-methyl and 6-thioxanthine. Only three of the single-Cys mutants of helix XII (V426C, N430C, and N443C) are sensitive to inactivation by N-ethylmaleimide. N430C is highly sensitive, with an IC50 of 10 microm, and is completely protected against inactivation in the presence of 2-thioxanthine, a high affinity substrate analogue. Other xanthine analogues are poorly bound by N430C, whereas replacement of Asn-430 with Thr inactivates the permease. The findings suggest that Ile-432 and Asn-430 of helix XII are crucial for purine uptake and affinity, and Asn-430 is probably at the vicinity of the binding site.en
heal.accesscampus-
heal.fullTextAvailabilityTRUE-
heal.identifier.primary10.1074/jbc.M800261200-
heal.identifier.secondaryhttp://www.ncbi.nlm.nih.gov/pubmed/18359771-
heal.identifier.secondaryhttp://www.jbc.org/content/283/20/13666.full.pdf-
heal.journalNameJ Biol Chemen
heal.journalTypepeer-reviewed-
heal.languageen-
heal.publicationDate2008-
heal.recordProviderΠανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικήςel
heal.typejournalArticle-
heal.type.elΆρθρο Περιοδικούel
heal.type.enJournal articleen

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