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Engineered occluded apo-intermediate of LacY.

Irina Smirnova1, Vladimir Kasho1, H Ronald Kaback2,3,4

  • 1Department of Physiology, University of California, Los Angeles, CA 90095-7327.

Proceedings of the National Academy of Sciences of the United States of America
|November 28, 2018
PubMed
Summary

Cross-linking lactose permease (LacY) in Escherichia coli stabilizes a sugar-binding occluded state. A nanobody reveals closed periplasmic access in mutants, suggesting a novel transport intermediate.

Keywords:
fluorescencelactose permeasemembrane transport proteinsnanobodiesstopped flow

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Transport

Background:

  • The lactose permease (LacY) from Escherichia coli is a model symporter.
  • It operates via an alternating access mechanism with multiple conformational states.
  • X-ray crystallography has primarily revealed inward- or outward-open structures.

Purpose of the Study:

  • To characterize novel conformational intermediates of LacY.
  • To investigate the accessibility of the sugar-binding site in different LacY states.
  • To elucidate the role of periplasmic and cytoplasmic access in LacY transport.

Main Methods:

  • Sugar-binding studies using engineered cysteine pairs in LacY.
  • Cross-linking experiments to stabilize specific conformers.
  • Utilizing a nanobody (Nb) to probe LacY structure and accessibility.
  • Assessing galactoside binding rates before and after disulfide bond reduction.

Main Results:

  • Paired-Cys mutations cross-linked across the cytoplasmic cavity stabilized an occluded LacY conformer.
  • This occluded state exhibited an inaccessible sugar-binding site.
  • A nanobody stabilizing a periplasmic-open state in wild-type LacY could not access the binding site in cross-linked mutants.
  • Nanobody binding and galactoside uptake were restored upon disulfide bond reduction, indicating periplasmic access.

Conclusions:

  • The cross-linked cytoplasmic mutants represent a stable, occluded apo-intermediate of LacY.
  • These findings provide structural insights into the transport cycle of LacY.
  • The periplasmic ends of the symporter helices appear tightly associated in the occluded state.