Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide

Abstract

The interaction between endotoxins-free lipid A and various lipopolysaccharide (LPS) chemotypes with different sugar chain lengths-and the polycationic peptides polymyxin B and polymyxin nonapeptide has been investigated by isothermal titration calorimetry between 20 and 50 degrees C. The results show a strong dependence of the titration curves on the phase state of the endotoxins. In the gel phase (<30 degrees C for LPS and <45 degrees C for lipid A), an endothermic reaction is observed, for which the driving force is an entropically driven endotoxin-polymyxin interaction, due to disruption of the ordered water structure and cation assembly in the lipid A backbone and adjacent molecules. In the liquid crystalline phase (>35 degrees C for LPS and >47 degrees C for lipid A) an exothermic reaction takes place, which is mainly due to the strong electrostatic interaction of the polymyxins with the negative charges of the endotoxins, i.e., the entropic change DeltaS is much lower than in the gel phase. For endotoxins with short sugar chains (lipid A, LPS Re, LPS Rc) the stoichiometry of the polymyxin binding corresponds to pure charge neutralization; for the compounds with longer sugar chains (LPS Ra, LPS S-form) this is no longer valid. This can be related to the lower susceptibility of the corresponding bacterial strains to antibiotics.

FIGURE 1

Chemical structures of (a) various rough mutant and S-form LPS from S. minnesota. Additional phosphates attached to the heptoses are present only for LPS Ra and S-form LPS. Abbreviations are: Hep, l-glycero-d-manno-heptopyranose; Glc, glucose; Gal, galactose. (b) Polymyxin B and its nonapeptide PMBN. DAB, diamino butyric acid.

FIGURE 2

Isothermal calorimetric titration of LPS Re (0.15 mM) from S. minnesota strain R595 with PMB (3 mM) at 20°C. For this, the LPS dispersion in the calorimetric cell was titrated every 5 min with 3 μl of PMB. The increase in the feedback power indicate an endothermic reaction.

FIGURE 3

Enthalpy change of the (a) LPS Re-PMB reaction versus [PMB]/[LPS Re] molar ratio in the temperature range 20–40°C, and (b) of the LPS Re-PMBN reaction versus [PMBN]/[LPS Re] molar ratio at temperatures 25 and 40°C.

FIGURE 4

Enthalpy change of the LPS Re-PMB reaction versus the [PMB]/[LPS Re] molar ratio at 24°C, in pure Hepes buffer (left) and in physiological saline (right).

FIGURE 5

Temperature dependence of ΔH, ΔG, and TΔS for the [LPS Re]/[PMB] binding. The presented data express only a tendency; a more precise treatment would imply more measuring points within each phase.

FIGURE 6

Synchrotron radiation small-angle x-ray diffraction patterns of the Mg ²⁺- salt form of LPS Re at 90% water content in the temperature range 20–70°C. The logarithm of the scattering intensity log I(s) is plotted versus the scattering vector s = 1/d (d-spacings in nm).

FIGURE 7

Differential scanning calorimetric heat capacity curves of mixtures of LPS and PMB (a) and LPS and PMBN (b) in various molar ratios. The specific excess heat capacity c_p,diff is plotted versus temperature. LPS concentration was 1 mg/ml (corresponding to 0.25 mM).

FIGURE 8

Enthalpy change of the lipid A-PMB reaction versus [PMB]/[lipid A] molar ratio in the temperature range 30–50°C. Binding saturation occurs at a molar ratio of 0.4:0.5.

FIGURE 9

Enthalpy change of the LPS Rc-PMB reaction versus [PMB]/[LPS Rc] molar ratio in the temperature range 25–45°C. Binding saturation occurs at a molar ratio of 1.0.

FIGURE 10

(a) Enthalpy change of the LPS Ra-PMB reaction versus [PMB]/[LPS Ra] molar ratio in the temperature range 20–40°C. (b) Enthalpy change of the LPS S-form-PMB reaction versus [PMB]/[LPS S-form] molar ratio in the temperature range 20–45°C. Binding saturation occurs at a molar ratio of 1.6:1.8 in the gel phase, and around 0.8 in the liquid crystalline phase.

FIGURE 11

Enthalpy change of the DOPC-PMB reaction versus [PMB]/[DOPC] ratio at 20 and 45°C.

FIGURE 12

Schematic model of PMB binding to the LPS aggregates in the gel phase (a) and liquid crystalline phase (b). In the gel phase, an ordered network of water and cations bridge neighboring LPS molecules, whereas in the liquid crystalline phase this network is disrupted. The diglucosamine backbone of lipid A exhibits an inclination of 40–50° with respect to the membrane plane according to previous results (Seydel et al., 2000).