Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport

Area of Science:

• Microbiology
• Structural Biology
• Biochemistry

Background:

• The Maintenance of Lipid Asymmetry (Mla) pathway is crucial for gram-negative bacteria, maintaining outer membrane integrity and contributing to virulence.
• This pathway involves multiple protein components, including the MlaA-OmpC complex, MlaC shuttle protein, and MlaFEDB ABC transporter, but the mechanism of phospholipid exchange remains unclear.
• Understanding phospholipid transfer is vital for elucidating the Mla pathway's function and the role of the MlaFEDB ATPase activity.

Purpose of the Study:

• To elucidate the mechanism of phospholipid transfer between components of the Mla pathway.
• To determine the structure of the E. coli MlaC-MlaD complex and identify key residues involved in phospholipid transport.

Main Methods:

• X-ray crystallography to determine the structure of the MlaC-MlaD complex.
• In vivo complementation assays to assess protein function.
• In vitro fluorescence-based transport assays to monitor phospholipid movement.
• Molecular dynamics simulations to analyze lipid trajectories.

Main Results:

• The structure of the E. coli MlaC in complex with the MlaD hexamer was determined in two distinct stoichiometries.
• Key residues essential for MlaCD function were identified, particularly within the MlaD β6-β7 loop.
• Evidence suggests phospholipids transfer between the C-terminal helices of the MlaD hexamer to reach its central pore.

Conclusions:

• This study provides the first structural insights into phospholipid transfer within the Mla pathway.
• The findings clarify the trajectory of glycerophospholipid (GPL) transfer between MlaC and MlaD, a critical step in maintaining bacterial membrane homeostasis.
• The MlaD β6-β7 loop is identified as a critical functional element for MlaCD-mediated lipid transport.