Structural determination of wild-type lactose permease

Area of Science:

• Structural Biology
• Membrane Protein Biochemistry
• Molecular Transport Mechanisms

Background:

• Lactose permease (LacY) is a crucial transporter protein in Escherichia coli, responsible for importing lactose into the cell.
• Understanding LacY's structure and mechanism is vital for deciphering secondary active transport systems.
• Previous studies provided structural insights into a mutant LacY, but the wild-type structure remained elusive.

Purpose of the Study:

• To determine the high-resolution x-ray structure of wild-type lactose permease (LacY) from Escherichia coli.
• To elucidate the structural basis for sugar and proton binding and translocation.
• To gain a deeper understanding of the transport mechanism and identify potential rate-limiting steps.

Main Methods:

• X-ray crystallography was employed to determine the three-dimensional structure of wild-type LacY.
• Crystallization conditions were optimized by manipulating phospholipid content.
• Structural analysis focused on the global fold, domain organization, and key functional residues.

Main Results:

• The x-ray structure of wild-type LacY was successfully determined, revealing a global fold consistent with previous mutant studies.
• LacY is organized into two six-helix bundles enclosing a central hydrophilic cavity.
• The cavity is open to the cytoplasmic side and contains residues critical for sugar and H+ binding.

Conclusions:

• The inward-facing conformation of LacY represents a low free-energy state.
• Sugar binding and/or an H+ electrochemical gradient likely promote opening towards the periplasmic side to initiate transport.
• The conformational change to the outward-facing state may be the rate-limiting step in the overall transport process.