Article

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.

Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece.
Journal of Biological Chemistry (impact factor: 4.77). 11/2010; 285(45):35011-20. DOI:10.1074/jbc.M110.170415 pp.35011-20
Source: PubMed

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).

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Keywords

67 single Cys mutants
 
bulky replacements
 
conserved 11-amino acid sequence
 
conserved motifs
 
Cys residues
 
Cys-scanning analysis
 
cytoplasmic end
 
Escherichia coli
 
functional mutant devoid
 
irreplaceable Glu-272
 
low levels
 
NAT signature motif
 
negligible expression
 
Pro-354 impair activity
 
purine binding site
 
sequences
 
Single Cys mutants V261C
 
transport mechanism cluster
 
ubiquitous nucleobase-ascorbate transporter
 
xanthine-specific homolog YgfO