Investigating Lipid Transporter Protein and Lipid Interactions Using Variable Temperature Electrospray Ionization,...

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Gram-negative bacteria possess an asymmetric outer membrane crucial for antibiotic resistance.
• The Mla (maintenance of lipid asymmetry) pathway, comprising six proteins, maintains this membrane structure.
• Understanding lipid transport is key to addressing antibiotic resistance.

Purpose of the Study:

• To investigate the lipid-binding preferences of the MlaC protein, a key component of the Mla pathway.
• To characterize the interaction between MlaC and its bound lipids.
• To determine how lipid binding affects MlaC protein structure and function.

Main Methods:

• Ultraviolet photodissociation (UVPD) was employed to identify and characterize endogenously bound lipids on MlaC.
• Collision cross-section measurements were used to monitor structural changes in MlaC upon lipid removal.
• Titrimetric experiments were conducted to evaluate MlaC's lipid selectivity.

Main Results:

• UVPD successfully identified and characterized lipid modifications bound to MlaC across different bacterial strains.
• Lipid binding was localized to MlaC residues 130-140, aligning with existing crystal structure data.
• MlaC undergoes unfolding before releasing its bound lipid, as indicated by collision cross-section measurements.
• MlaC exhibits selectivity for lipids with a maximum of two acyl chains, including sphingolipids, glycerophospholipids, and fatty acids.

Conclusions:

• MlaC plays a critical role in transporting lipids within the periplasm of Gram-negative bacteria.
• The study elucidates MlaC's lipid-binding site and preferences, contributing to understanding membrane asymmetry maintenance.
• The findings provide insights into the structural dynamics of MlaC during lipid transport and release, potentially informing strategies against antibiotic resistance.