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An Asymmetric Conformational Change in LacY
Irina Smirnova1, Vladimir Kasho1, Xiaoxu Jiang1
1Department of Physiology, ‡Department of Microbiology, Immunology & Molecular Genetics, and §Molecular Biology Institute, University of California Los Angeles , Los Angeles, California 90095-7327, United States.
Biochemistry
|March 17, 2017
Summary
The lactose permease (LacY) in E. coli uses alternating access. Blocking the periplasmic side prevents sugar binding, but opening it restores binding, revealing asymmetric conformational control.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- The lactose permease (LacY) of Escherichia coli facilitates galactoside and proton symport across the cell membrane.
- This process relies on alternating access of internal binding sites to the periplasmic and cytoplasmic sides.
- Understanding LacY's conformational changes is key to elucidating sugar/H+ symport mechanisms.
Purpose of the Study:
- To investigate the role of periplasmic and cytoplasmic cavity accessibility in LacY-mediated galactoside transport.
- To determine how conformational changes influence sugar binding kinetics and accessibility.
- To explore the mechanism of asymmetrical conformational transitions in symporter function.
Main Methods:
- Reconstitution of purified lactose permease (LacY) into proteoliposomes.
- Site-directed mutagenesis to introduce paired cysteine residues for cross-linking.
- Disulfide bond formation and reduction to control periplasmic cavity accessibility.
- Nanobody stabilization of specific LacY conformations.
- Galactoside binding assays and nonlinear stopped-flow kinetics analysis.
Main Results:
- Cross-linking LacY to seal the periplasmic cavity abolished periplasmic galactoside binding.
- Reduction of the disulfide bond restored periplasmic cavity opening and galactoside binding.
- Nanobodies stabilized a periplasmic-open conformation only after disulfide bond reduction.
- Solubilized, cross-linked LacY showed cytoplasmic galactoside binding, indicating a patent cytoplasmic cavity.
- Cytoplasmic sugar binding exhibited nonlinear kinetics, suggesting a two-step binding process involving a conformational change.
Conclusions:
- Periplasmic cavity accessibility is essential for galactoside binding from the periplasmic side.
- The lactose permease undergoes asymmetrical conformational transitions that control access to the binding site.
- Spontaneous opening and closing of the cytoplasmic cavity in a sealed periplasmic state suggest directional control of transport.
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