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Molecular Basis for the Activation of Pseudomonas aeruginosa MsbA by Divalent Metals
Jixing Lyu1, Hanieh Bahramimoghaddam1, Tianqi Zhang1
1Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
Abstract:
Proteins involved in the biogenesis of lipopolysaccharide (LPS), a lipid exclusive to Gram-negative bacteria, are promising candidates for drug discovery. Specifically, the ABC transporter MsbA plays a crucial role in translocating an LPS precursor from the cytoplasmic to the periplasmic facing leaflet of the inner membrane, and small molecules that inhibit its function exhibit bactericidal activity. Here, we use native mass spectrometry (MS) to determine lipid binding affinities of MsbA from P. aeruginosa (PaMsbA), a Gram-negative bacteria associated with hospital-acquired infections, in different conformations. Unlike the transporter from E. coli, we show that the ATPase activity of PaMsbA is stimulated by Zn2+, Ni2+, and Mn2+ and successfully trapping the protein with vanadate requires one of these metal ions. We also present cryogenic-electron microscopy structures of PaMsbA in occluded and open outward-facing conformations determined to resolutions of 2.58 and 2.44 Å, respectively. The structures reveal a triad of histidine residues, and mutation of these residues abolishes Zn2+ binding and stimulation of PaMsbA activity by metal ions. Together, our studies provide insight into the structure of PaMsbA and its lipid binding preferences and reveal that a subset of divalent metals stimulates its ATPase activity.
Insights
The ABC transporter MsbA is crucial for Gram-negative bacteria. This study reveals how metal ions like zinc stimulate its activity and provides structural insights into PaMsbA, a potential drug target for infections.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Lipopolysaccharide (LPS) biogenesis in Gram-negative bacteria involves essential proteins like the ABC transporter MsbA.
- Inhibiting MsbA function offers a potential strategy for developing new antibacterial drugs.
- Pseudomonas aeruginosa (Paeruginosa) is a significant cause of hospital-acquired infections.
Purpose of the Study:
- To investigate the lipid binding affinities and conformational states of Paeruginosa MsbA (PaMsbA).
- To elucidate the role of divalent metal ions in stimulating PaMsbA's ATPase activity.
- To determine the structural basis for metal ion interaction and activity modulation in PaMsbA.
Main Methods:
- Native mass spectrometry (MS) was employed to assess lipid binding affinities of PaMsbA.
- Cryo-electron microscopy (cryo-EM) was used to resolve PaMsbA structures in different conformations.
- Site-directed mutagenesis was performed to investigate the role of key histidine residues.
Main Results:
- PaMsbA's ATPase activity is stimulated by specific divalent metal ions (Zn2+, Ni2+, Mn2+), unlike E. coli MsbA.
- Cryo-EM structures revealed occluded and open outward-facing conformations of PaMsbA.
- A triad of histidine residues was identified as crucial for metal ion binding and activity stimulation.
Conclusions:
- Divalent metal ions significantly influence PaMsbA's ATPase activity and conformational states.
- Structural insights into PaMsbA, including metal ion interactions, provide a foundation for drug discovery targeting Gram-negative bacteria.
- Understanding PaMsbA's mechanism is key to developing novel therapeutics against P. aeruginosa infections.
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