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The role of transmembrane segment TM3 in the xanthine permease XanQ of Escherichia coli.

ABSTRACT: The xanthine permease XanQ of Escherichia coli is used as a study prototype for function-structure analysis of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family. Our previous mutagenesis study of polar residues of XanQ has shown that Asn-93 at the middle of putative TM3 is a determinant of substrate affinity and specificity. To study the role of TM3 in detail we employed Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence 79-107 (YGIVGSGLLSIQSVNFSFVTVMIALGSSM) including TM3 (underlined) and flanking sequences was replaced individually with Cys. Of 29 single-Cys mutants, 20 accumulate xanthine to 40-110% of the steady state observed with C-less, six (S88C, F94C, A102C, G104C, S106C) accumulate to low levels (10-30%) and three (G83C, G85C, N93C) are inactive. Extensive mutagenesis reveals that Gly-83 and, to a lesser extent, Gly-85, are crucial for expression in the membrane. Replacements of Asn-93 disrupt affinity (Thr) or permit recognition of 8-methylxanthine which is not a wild-type ligand (Ala, Ser, Asp) and utilization of uric acid which is not a wild-type substrate (Ala, Ser). Replacements of Phe-94 impair affinity for 2-thio and 6-thioxanthine (Tyr) or 3-methylxanthine (Ile). Single-Cys mutants S84C, L86C, L87C, and S95C are highly sensitive to inactivation by N-ethylmaleimide. Our data reveal that key residues of TM3 cluster in two conserved sequence motifs, (83)GSGLL(87) and (93)NFS(95), and highlight the importance of Asn-93 and Phe-94 in substrate recognition and specificity; these findings are supported by structural modeling on the recently described x-ray structure of the uracil-transporting homolog UraA.

SUBMITTER: Karena E

PROVIDER: S-EPMC3234782 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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The role of transmembrane segment TM3 in the xanthine permease XanQ of Escherichia coli.

Karena Ekaterini E Frillingos Stathis S

The Journal of biological chemistry 20110914 45

The xanthine permease XanQ of Escherichia coli is used as a study prototype for function-structure analysis of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family. Our previous mutagenesis study of polar residues of XanQ has shown that Asn-93 at the middle of putative TM3 is a determinant of substrate affinity and specificity. To study the role of TM3 in detail we employed Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue i ...[more]

PMID: 21917919

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Project description:Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.

Dataset Information

The role of transmembrane segment TM3 in the xanthine permease XanQ of Escherichia coli.

Publications

The role of transmembrane segment TM3 in the xanthine permease XanQ of Escherichia coli.

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