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Updated: Aug 1, 2025
Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro
Published on: January 11, 2017
Protein-protein interactions in the Mla lipid transport system probed by computational structure prediction and deep
Mark R MacRae1, Dhenesh Puvanendran1, Max A B Haase1
1Department of Cell Biology, New York University School of Medicine, New York, New York, USA.
Abstract:
The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer that protects the cell from external stressors, such as antibiotics. The Mla transport system is implicated in the Maintenance of OM Lipid Asymmetry by mediating retrograde phospholipid transport across the cell envelope. Mla uses a shuttle-like mechanism to move lipids between the MlaFEDB inner membrane complex and the MlaA-OmpF/C OM complex, via a periplasmic lipid-binding protein, MlaC. MlaC binds to MlaD and MlaA, but the underlying protein-protein interactions that facilitate lipid transfer are not well understood. Here, we take an unbiased deep mutational scanning approach to map the fitness landscape of MlaC from Escherichia coli, which provides insights into important functional sites. Combining this analysis with AlphaFold2 structure predictions and binding experiments, we map the MlaC-MlaA and MlaC-MlaD protein-protein interfaces. Our results suggest that the MlaD and MlaA binding surfaces on MlaC overlap to a large extent, leading to a model in which MlaC can only bind one of these proteins at a time. Low-resolution cryo-electron microscopy (cryo-EM) maps of MlaC bound to MlaFEDB suggest that at least two MlaC molecules can bind to MlaD at once, in a conformation consistent with AlphaFold2 predictions. These data lead us to a model for MlaC interaction with its binding partners and insights into lipid transfer steps that underlie phospholipid transport between the bacterial inner and OMs.
Insights
The Mla transport system maintains bacterial outer membrane lipid asymmetry. This study reveals how MlaC protein interacts with MlaA and MlaD, clarifying lipid transport mechanisms essential for cell envelope integrity.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Gram-negative bacteria possess an outer membrane (OM) crucial for protection against environmental threats like antibiotics.
- The Mla (Maintenance of OM Lipid Asymmetry) transport system facilitates phospholipid transport, maintaining OM integrity.
- The Mla system involves inner membrane (IM) and OM complexes, linked by the periplasmic lipid-binding protein MlaC.
Purpose of the Study:
- To elucidate the protein-protein interactions between MlaC and its binding partners, MlaA and MlaD.
- To map the functional sites of MlaC involved in lipid transfer within the Mla transport system.
- To develop a mechanistic model for MlaC's role in retrograde phospholipid transport.
Main Methods:
- Deep mutational scanning of MlaC from Escherichia coli to identify functionally important residues.
- AlphaFold2 structure prediction to model protein complexes.
- Binding experiments and cryo-electron microscopy (cryo-EM) to analyze MlaC interactions with MlaA, MlaD, and the MlaFEDB complex.
Main Results:
- Deep mutational scanning identified key functional sites on MlaC.
- MlaC's binding surfaces for MlaA and MlaD significantly overlap, suggesting sequential binding.
- Cryo-EM data indicate MlaC can bind to the MlaD component of the MlaFEDB complex, potentially with multiple MlaC molecules.
Conclusions:
- MlaC likely binds MlaA and MlaD sequentially, not simultaneously, facilitating lipid transfer.
- The study provides a refined model for MlaC's interaction dynamics within the Mla transport system.
- These findings offer insights into the molecular mechanisms of phospholipid transport and OM homeostasis in bacteria.
