title: Cysteine-scanning analysis of helices TM8, TM9a, and TM9b and intervening loops in the YgfO xanthine permease: a carboxyl group is essential at ASP-276.
abstract: Bacterial and fungal members of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cys-scanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences (259)FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV(314) and (342)TIAVMLVILGLFP(354) including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35-140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10-20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (K(i) 79 μm) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC(50) 15 μm) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXGDXXAT) and in TM9a (GXXXDG).
authors.affiliation: Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Mermelekas
authors.forename: Ekaterini
authors.initials: E
authors.lastname: Georgopoulou
authors.forename: Alexander
authors.initials: A
authors.lastname: Kallis
authors.forename: Maria
authors.initials: M
authors.lastname: Botou
authors.forename: Vassilios
authors.initials: V
authors.lastname: Vlantos
authors.forename: Stathis
authors.initials: S
authors.lastname: Frillingos
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D033702:Nucleobase Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D033702:Nucleobase Transport Proteins; C529797:YgfO protein, E coli; D003545:Cysteine
references.citation: J Biol Chem. 2008 May 16;283(20):13666-78
references.pmid: 18359771
references.citation: Eur J Biochem. 1980;108(1):223-31
references.pmid: 6250828
references.citation: Biochemistry. 2001 Jan 23;40(3):769-76
references.pmid: 11170394
references.citation: Mol Biosyst. 2008 May;4(5):404-16
references.pmid: 18414738
references.citation: J Mol Biol. 2005 Jul 15;350(3):499-513
references.pmid: 15953615
references.citation: Nature. 2004 Oct 14;431(7010):811-8
references.pmid: 15483603
references.citation: J Biol Chem. 2006 Dec 29;281(52):39881-90
references.pmid: 17077086
references.citation: J Mol Biol. 2010 Apr 16;397(5):1132-43
references.pmid: 20188741
references.citation: J Mol Biol. 2008 Oct 24;382(5):1121-35
references.pmid: 18718842
references.citation: J Biol Chem. 2010 Feb 26;285(9):6522-31
references.pmid: 20042597
references.citation: Gene. 1990 Nov 30;96(1):23-8
references.pmid: 2265755
references.citation: J Biol Chem. 2009 Sep 4;284(36):24257-68
references.pmid: 19581302
references.citation: J Mol Biol. 2005 Sep 23;352(3):489-94
references.pmid: 16120447
references.citation: Exp Cell Res. 2009 Aug 1;315(13):2312-21
references.pmid: 19379732
references.citation: J Biol Chem. 2010 Jun 18;285(25):19422-33
references.pmid: 20406814
references.citation: J Mol Biol. 2002 Jan 25;315(4):561-71
references.pmid: 11812130
references.citation: Mol Membr Biol. 2005 May-Jun;22(3):251-61
references.pmid: 16096267
references.citation: Nature. 1999 May 6;399(6731):70-5
references.pmid: 10331392
references.citation: Proc Natl Acad Sci U S A. 2000 Sep 26;97(20):10729-32
references.pmid: 10984523
references.citation: Biochemistry. 1996 Aug 6;35(31):10166-71
references.pmid: 8756481
references.citation: J Biol Chem. 2001 Jun 8;276(23):20330-9
references.pmid: 11278375
references.citation: Acta Biochim Pol. 2004;51(2):493-531
references.pmid: 15218545
references.citation: EMBO J. 1998 Jul 15;17(14):3827-37
references.pmid: 9670000
references.citation: Proc Natl Acad Sci U S A. 2000 May 23;97(11):5796-801
references.pmid: 10823938