Substrate-induced changes in the structural properties of LacY.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Lactose permease (LacY) from Escherichia coli is a model transporter in the major facilitator superfamily.
• LacY facilitates the coupled transport of galactopyranoside and H+ across the cell membrane.
• Large conformational changes are essential for LacY's alternating access mechanism, but the triggering events remain unclear.

Purpose of the Study:

• To investigate how substrate binding induces conformational changes in LacY.
• To elucidate the role of mechanical properties in LacY's transport mechanism.

Main Methods:

• Dynamic single-molecule force spectroscopy was employed.
• Mechanical, kinetic, and energetic properties of LacY were analyzed.

Main Results:

• Galactoside binding significantly alters the N-terminal 6-helix bundle of LacY, particularly helix V.
• The N-terminal domain transitions from a mechanically brittle to a flexible state upon substrate binding, with increased stability and lifetime.
• The C-terminal domain remained largely unaffected by galactoside binding.
• A Cys154→Gly mutant showed no changes in properties upon sugar binding, indicating altered intramolecular interactions.

Conclusions:

• Substrate binding to LacY tunes the mechanical properties of its N-terminal domain to initiate symport.
• The findings support a model where substrate binding drives alternating conformational states in LacY.
• This study provides insights into the kinetic, energetic, and mechanical underpinnings of LacY function.