Biophysical characterisation of lysozyme binding to LPS Re and lipid A

Abstract

The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide (LPS) Re and to its lipid moiety lipid A, the 'endotoxic principle' of LPS, was investigated using biophysical techniques. The β↔ A gel to liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X-ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl-chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stochiometry of endotoxin and lysozyme is a 3 :1 molar ratio for both LPS Re and lipid A, indicating a dominant binding of lysozyme to the lipid A-phosphates. This could be confirmed by the analysis of a phosphate vibration and by the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly A helix to more unordered structures takes place, while the residual β-sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.

Biophysical Characterization of Endotoxin Inactivation by NK-2, an Antimicrobial Peptide Derived from Mammalian NK-Lysin

“…It was previously found that the conical shape of the lipid A part of the LPS molecule is necessary to exhibit endotoxic activity in a biological system (16,38). This lamellarization of the endotoxin aggregates has also been observed for other proteins and polypeptides that inhibit the cell stimulation activity of LPS, such as lactoferrin (14), high-density lipoprotein (13), lysozyme (15), and polymyxin B (17). The interaction of LPS with LBP and CD14 may thus be directly influenced as a consequence of this structural change.…”

Structural Basis for Interactions between Lung Surfactant Protein C and Bacterial Lipopolysaccharide

“…This is particularly the case with polymyxin B, lysozyme, the limulus anti-LPS factor, and with the two structurally related mammalian proteins that share high affinity binding to LPS: bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein. For example, polymyxin B and lysozyme bind electrostatically to the phosphate groups of lipid A, as shown by analysis of phosphate vibration (42). Concerning LPS-binding protein and bactericidal/permeability-increasing protein, the interactions of these proteins with LPS seems to be a complex multisystem process involving, on one hand, the lipid-binding pockets of these molecules (which can accommodate the apolar moiety of LPS but with little specificity) and, on the other hand, positively charged groups (between residues 90 and 103) (43) leading to electrostatic interactions with phosphorylated sugar groups of LPS (44).…”

Liquid Crystal Biosensors: New Approaches

“…It is important to understand the interaction of proteins with endotoxins as it led to divergent effects on LPSinduced responses (Tesh and Morrison, 1988; Roth and Kaca, 1994; Machnicki et al, 1993;Yu and Wright 1996;Brandenburg et al, 1998), which serve as a basis for many clinical and therapeutic applications. Inspired by this, we report a simple but useful advance in the design of an experimental system that enables a quick, quantitative, and label-free detection of LPSbiomolecular interactions (Das et al, 2015).…”