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The substrate-binding site in the lactose permease of Escherichia coli
1Howard Hughes Medical Institute, Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Abstract:
Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 --> Cys was also studied. In marked contrast to Val-331 --> Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 --> Ala/Val-331 --> Cys, Arg-144 --> Ala/Val-331 --> Cys, or Arg-144 --> Lys/Val-331 --> Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 --> Ala or Arg-144 --> Ala and, to a lesser extent, Arg-144 --> Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I-. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H+ translocation pathway at the interface between helices IX and X.
Insights
Glutamate-126 and Arginine-144 in lactose permease are crucial for substrate binding, forming a charge pair essential for normal function. Mutations disrupt ligand protection and alter protein conformation, impacting transport pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- Lactose permease facilitates sugar transport across membranes.
- Understanding the substrate-binding site is key to elucidating transport mechanisms.
Purpose of the Study:
- Investigate the roles of Glutamate-126 (Glu-126) and Arginine-144 (Arg-144) in lactose permease function.
- Determine the structural and functional significance of these residues in substrate binding and translocation.
Main Methods:
- Site-directed mutagenesis to create specific amino acid substitutions (e.g., Glu-126 to Ala, Arg-144 to Ala).
- N-ethylmaleimide labeling to assess cysteine reactivity at position 148.
- Site-directed fluorescence labeling with MIANS to probe conformational changes.
Main Results:
- Mutations at Glu-126 and Arg-144 significantly reduce Cys-148 reactivity, indicating they form a charge pair.
- Ligand binding fails to protect Cys-148 from alkylation in these mutants.
- Fluorescence studies reveal altered conformational dynamics and fluorophore accessibility in mutants.
Conclusions:
- Glu-126 and Arg-144 are essential and irreplaceable for substrate binding in lactose permease.
- These residues likely form a charge pair critical for maintaining the integrity of the substrate-binding site.
- Conformational changes in the substrate pathway are transmitted to the H+ translocation pathway.