Interactions of lipopolysaccharide and polymyxin studied by NMR spectroscopy
Jiri Mares1, Sowmini Kumaran, Marina Gobbo
1Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, Zurich CH 8057, Switzerland.
Abstract:
In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region.
Insights
Antimicrobial peptides like polymyxins combat antibiotic-resistant bacteria by binding to lipopolysaccharide (LPS). This study reveals the precise interaction mechanism between polymyxins and LPS using advanced spectroscopy and modeling.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Increasing antibiotic resistance necessitates novel therapeutic strategies.
- Antimicrobial peptides, such as polymyxins, show promise against Gram-negative bacteria.
- Polymyxins target the outer membrane component, lipopolysaccharide (LPS).
Purpose of the Study:
- To elucidate the interaction mechanism between polymyxins (PMX-B, -E, -M) and Escherichia coli lipopolysaccharide (LPS).
- To develop an efficient LPS purification method.
- To model the complex formed between LPS and PMX-B.
Main Methods:
- Developed an efficient LPS purification protocol using reversed-phase high-performance liquid chromatography.
- Utilized heteronuclear NMR spectroscopy (chemical shift mapping, isotope-filtered NOESY) to study LPS-polymyxin interactions in a dodecylphosphocholine micelle model.
- Performed molecular dynamics calculations to model the LPS-PMX-B complex using NMR-derived restraints.
Main Results:
- Established an efficient method for purifying biosynthetically produced LPS.
- NMR data revealed the specific binding mode of polymyxins to LPS.
- Molecular dynamics modeling showed PMX macrocycle interaction with the LPS phosphate group, with side chain contacts and hydrophobic penetration.
Conclusions:
- Detailed the molecular interactions between polymyxins and LPS, crucial for understanding their antimicrobial activity.
- The findings provide a structural basis for the mechanism of action of polymyxins against Gram-negative bacteria.
- This research contributes to the development of new antimicrobial agents to combat resistant bacterial strains.
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