Interpretation of biological activity data of bacterial endotoxins by simple molecular models of mechanism of action

Lipid A moiety has been identified as the bioactive component of bacterial endotoxins (lipopolysaccharides). However, the molecular mechanism of biolo gical activity of lipid A is still not fully understood. This paper contrib utes to understanding of the molecular mechanism of action of bacterial end otoxins by comparing molecular modelling results for two possible mechanism s with the underlying experimental data. Mechanisms of action involving spe cific binding of lipid A to a protein receptor as well as nonspecific inter calation into phospholipid membrane of a host cell were modelled and analys ed. As the cellular receptor for endotoxin has not been identified, a model of a peptidic pseudoreceptor was proposed, based on molecular structure, s ymmetry of the lipid A moiety and the observed character of endotoxin-bindi ng sites in proteins. We have studied the monomeric form of lipid A from Es cherichia coli and its seven synthetic analogues with varying numbers of ph osphate groups and correlated them with known biological activities determi ned by the Limulus assay. Gibbs free energies associated with the interacti on of lipid A with the pseudoreceptor model and intercalation into phosphol ipid membrane calculated by molecular mechanics and molecular dynamics meth ods were used to compare the two possible mechanisms of action. The results suggest that specific binding of lipid A analogues to the peptidic pseudor eceptor carrying an amphipathic cationic binding pattern BHPHB (B, basic; H , hydrophobic; P, polar residue, respectively) is energetically more favour able than intercalation into the phospholipid membrane. Zn addition, bindin g affinities of lipid A analogues to the best minimum binding sequence KFSF K of the pseudoreceptor correlated with the experimental Limulus activity p arameter. This correlation enabled us to rationalize the observed relations hip between the number and position of the phosphate groups in the lipid A moiety and its biological activity in terms of specific ligand-receptor int eractions. If lipid A-receptor interaction involves formation of phosphate- ammonium ion-pair(s) with cationic amino-acid residues, the specific mechan ism of action was fully consistent with the underlying experimental data. A s a consequence, recognition of lipid A variants by an amphipathic binding sequence BHPHB of a host-cell protein receptor might represent the initial and/or rate-determining molecular event of the mechanism of action of lipid A (or endotoxin). The insight into the molecular mechanism of action and t he structure of the lipid A-binding pattern have potential implications for rational drug design strategies of endotoxin-neutralizing agents or bindin g factors.