PMID:1438245
| Citation |
Sahin-Tóth, M, Dunten, RL, Gonzalez, A and Kaback, HR (1992) Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 89:10547-51 |
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| Abstract |
Using a lactose permease mutant devoid of Cys residue ("C-less permease"), we systematically replaced putative intramembrane charged residues with Cys. Individual replacements for Asp-237, Asp-240, Glu-269, Arg-302, Lys-319, His-322, Glu-325, or Lys-358 abolish active lactose transport. When Asp-237 and Lys-358 are simultaneously replaced with Cys and/or Ala, however, high activity is observed. Therefore, when either Asp-237 or Lys-358 is replaced with a neutral residue, leaving an unpaired charge, the permease is inactivated, but neutral replacement of both residues yields active permease [King, S. C., Hansen, C. L. & Wilson, T. H. (1991) Biochim. Biophys. Acta 1062, 177-186]. Remarkably, moreover, when Asp-237 is interchanged with Lys-358, high activity is observed. The observations provide a strong indication that Asp-237 and Lys-358 interact to form a salt bridge. In addition, the data demonstrate that neither residue nor the salt bridge plays an important role in the transport mechanism. Thirteen additional double mutants were constructed in which a negative and a positively charged residue were replaced with Cys. Only Asp-240-->Cys/Lys-319-->Cys exhibits significant activity, accumulating lactose to 25-30% of the steady state observed with C-less permease. Replacing either Asp-240 or Lys-319 individually with Ala also inactivates the permease, but double mutants with neutral substitutions (Cys and/or Ala) at both positions exhibit essentially the same activity as Asp-240-->Cys/Lys-319-->Cys. In marked contrast to Asp-237 and Lys-358, interchanging Asp-240 and Lys-319 abolishes active lactose transport. The results demonstrate that Asp-240 and Lys-319, like Asp-237 and Lys-358, interact functionally and may form a salt bridge. However, the interaction between Asp-240 and Lys-319 is clearly more complex than the interaction between Asp-237 and Lys-358. In any event, the findings suggest that putative transmembrane helix VII lies next to helices X and XI in the tertiary structure of lactose permease. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Biological Transport, Active; Cell Membrane/enzymology; DNA, Bacterial/genetics; DNA, Bacterial/isolation & purification; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli Proteins; Kinetics; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Models, Structural; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Plasmids; Protein Structure, Secondary; Symporters |
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