Cryo-EM structure of MsbA in saposin-lipid nanoparticles (Salipro) provides insights into nucleotide coordination
Dominique-Maurice Kehlenbeck1,2,3,4, Daouda A K Traore3,4,5,6, Inokentijs Josts1,2
1The Hamburg Advanced Research Center for Bioorganic Chemistry (HARBOR), Germany.
Abstract:
The ATP-binding cassette transporter MsbA is a lipid flippase, translocating lipid A, glycolipids, and lipopolysaccharides from the inner to the outer leaflet of the inner membrane of Gram-negative bacteria. It has been used as a model system for time-resolved structural studies as several MsbA structures in different states and reconstitution systems (detergent/nanodiscs/peptidiscs) are available. However, due to the limited resolution of the available structures, detailed structural information on the bound nucleotides has remained elusive. Here, we have reconstituted MsbA in saposin A-lipoprotein nanoparticles (Salipro) and determined the structure of ADP-vanadate-bound MsbA by single-particle cryo-electron microscopy to 3.5 Å resolution. This procedure has resulted in significantly improved resolution and enabled us to model all side chains and visualise detailed ADP-vanadate interactions in the nucleotide-binding domains. The approach may be applicable to other dynamic membrane proteins.
Insights
Researchers visualized the nucleotide-binding site of the MsbA transporter using cryo-EM. This improved resolution reveals detailed interactions of ADP-vanadate, advancing understanding of lipid flippase mechanisms in bacteria.
Area of Science:
- Structural Biology
- Biochemistry
- Microbiology
Background:
- ATP-binding cassette transporter MsbA functions as a lipid flippase in Gram-negative bacteria.
- MsbA is a model for time-resolved structural studies, with existing structures in various states and reconstitution systems.
- Previous structural studies lacked the resolution to detail bound nucleotides in MsbA.
Purpose of the Study:
- To determine the high-resolution structure of ADP-vanadate-bound MsbA.
- To visualize detailed nucleotide interactions within MsbA's binding domains.
- To establish a method applicable to other dynamic membrane proteins.
Main Methods:
- Reconstitution of MsbA in saposin A-lipoprotein nanoparticles (Salipro).
- Single-particle cryo-electron microscopy (cryo-EM) for structure determination.
- High-resolution structural analysis to model nucleotide interactions.
Main Results:
- Determined the structure of ADP-vanadate-bound MsbA at 3.5 Å resolution.
- Achieved significantly improved resolution compared to previous studies.
- Successfully modeled all side chains and visualized detailed ADP-vanadate interactions.
Conclusions:
- The Salipro reconstitution method enhances structural resolution for MsbA.
- Detailed visualization of nucleotide binding provides insights into MsbA's mechanism.
- The employed approach shows potential for studying other dynamic membrane proteins.
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