Helix dynamics in LacY: helices II and IV

Area of Science:

• Biochemistry
• Structural Biology
• Membrane Transport

Background:

• Lactose permease (LacY) from Escherichia coli facilitates lactose and proton symport across the cell membrane.
• Previous studies highlighted the importance of Asp68 in helix II for LacY conformational changes during transport.
• A model suggests alternating access of binding sites to both sides of the membrane drives symport.

Purpose of the Study:

• To investigate the role of Asp68 in the symport mechanism of lactose permease.
• To elucidate the structural and functional consequences of Asp68 mutations on LacY activity.
• To refine the model of LacY-mediated symport.

Main Methods:

• Site-directed mutagenesis of lactose permease (LacY), specifically targeting Asp68.
• Biochemical assays to assess active transport and ligand binding.
• X-ray crystallography to determine structures of wild-type and mutant LacY.
• Site-directed alkylation studies.

Main Results:

• Mutations at Asp68, particularly with glutamate, abolished active transport while retaining galactopyranoside binding.
• X-ray structures revealed Asp68 and Lys131 (helix IV) are in proximity.
• Asp68 mutants showed sugar binding-induced closure of the cytoplasmic cavity but reduced opening of the periplasmic pathway.
• Double mutants involving Cys at positions 68 and 131 cross-linked efficiently, suggesting helix proximity.

Conclusions:

• Replacement of Asp68 disrupts a critical conformational transition involving helices II and IV, essential for opening the periplasmic cavity.
• This disruption blocks the symport mechanism by preventing the necessary opening of the periplasmic pathway.
• Functional coupling between helices II/IV and helices VIII/X is suggested in the transport mechanism.