Role of intramembrane polar residues in the YgfO xanthine permease: HIS-31 and ASN-93 are crucial for affinity and specificity, and ASP-304 and GLU-272 are irreplaceable.
Using the YgfO xanthine permease of Escherichia coli as a bacterial model for the study of the evolutionarily ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family, we performed a systematic Cys-scanning and site-directed mutagenesis of 14 putatively charged (Asp, Glu, His, Lys, or Arg) and 7 highly polar (Gln or Asn) residues that are predicted to lie in transmembrane helices (TMs). Of 21 single-Cys mutants engineered in the background of a functional YgfO devoid of Cys residues (C-less), only four are inactive or have marginal activity (H31C, N93C, E272C, D304C). The 4 residues are conserved throughout the family in TM1 (His-31), TM3 (Asn-93/Ser/Thr), TM8 (Glu-272), and putative TM9a (Asp-304/Asn/Glu). Extensive site-directed mutagenesis in wild-type background showed that H31N and H31Q have high activity and affinity for xanthine but H31Q recognizes novel purine bases and analogues, whereas H31C and H31L have impaired affinity for xanthine and analogues, and H31K or H31R impairs expression in the membrane. N93S and N93A are highly active but more promiscuous for recognition of analogues at the imidazole moiety of substrate, N93D has low activity, N93T has low affinity for xanthine or analogues, and N93Q or N93C is inactive. All mutants replacing Glu-272 or Asp-304, including E272D, E272Q, D304E, and D304N, are inactive, although expressed to high levels in the membrane. Finally, one of the 17 assayable single-Cys mutants, Q258C, was sensitive to inactivation by N-ethylmaleimide. The findings suggest that polar residues important for the function of YgfO cluster in TMs 1, 3, 8 and 9a.
Karena
Ekaterini
E
Laboratory of Biological Chemistry, University of Ioannina Medical School, 45110 Ioannina, Greece.
Frillingos
Stathis
S
D019943:Amino Acid Substitution; D001216:Asparagine / Q000235:genetics / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D020125:Mutation, Missense; D033702:Nucleobase Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D019820:Xanthine / Q000378:metabolism
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; C529797:YgfO protein, E coli; D019820:Xanthine; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D001216:Asparagine