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Galactoside-binding site in LacY.

Xiaoxu Jiang1, Maria Katerina R Villafuerte, Magnus Andersson

  • 1Department of Physiology and Department of Microbiology, Immunology and Molecular Genetics, Molecular Biology Institute, University of California at Los Angeles , Los Angeles, California 90095-7327, United States.

Biochemistry
|February 14, 2014
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Summary
This summary is machine-generated.

Lactose permease (LacY) binding site identification was advanced by molecular dynamics and mutagenesis. Asn272 and Gly268 are key residues in galactoside binding, clarifying LacY

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The precise identification of ligand-binding residues in lactose permease (LacY) remains challenging despite available X-ray crystal structures.
  • Previous studies suggested potential roles for residues Asn272, Gly268, and Val264 in galactoside binding based on site-directed alkylation data.

Purpose of the Study:

  • To precisely identify the specific amino acid residues involved in galactoside binding within lactose permease (LacY).
  • To elucidate the functional contribution of Asn272, Gly268, and Val264 to substrate binding affinity and specificity.

Main Methods:

  • Molecular dynamics simulations were employed to model the interaction between lactose permease and a docked lactose analogue.
  • Site-directed mutagenesis was performed on Asn272, Gly268, and Val264 to assess their individual contributions to substrate binding.
  • Binding affinities (KD) of wild-type and mutant LacY proteins were measured to quantify the effects of mutations.

Main Results:

  • Molecular dynamics simulations indicated that Asn272 is positioned favorably for interaction with the galactopyranosyl ring, suggesting a crucial role in binding.
  • Mutagenesis studies revealed that replacements of Asn272 significantly reduced or abolished substrate binding, except for the Asn272 → Gln mutant.
  • Mutant Gly268 → Ala showed an 8-fold decrease in binding affinity, while mutant Val264 → Ala exhibited minimal changes, indicating a moderate role for Gly268 and a negligible role for Val264.

Conclusions:

  • Asn272 is a critical residue for galactoside binding in lactose permease (LacY).
  • Gly268 may also contribute to the galactoside-binding site, albeit to a lesser extent than Asn272.
  • These findings provide crucial insights into the molecular mechanism of lactose transport by LacY.