Insights into the initial stages of lipid-mediated haemozoin nucleation

Abstract

Lipid-mediated haemozoin (Hz) formation was investigated using experimental and computational techniques. Glycerolipids (predominantly diacylglycerols), not phospholipids, were found entrapped by Hz crystals isolated from Plasmodium falciparum and consequently are proposed to nucleate Hz crystals in vivo. The initial stages of Hz formation were investigated using molecular dynamics simulations which involved modelling ferrihaem species pertinent to Hz formation (π–π and μ-propionato dimers) at lipid–water interfaces comprising a representative glycerolipid 1,2-dioleoyl-sn-glycerol (DOG) or phospholipid 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Simulations showed that π–π dimers partition into both lipids and remain near the hydrated lipid–water interface. Dimer aggregation, which was only observed in DOG, is proposed to be crucial for nucleating Hz. Indeed, β-haematin (synthetic Hz) production was found experimentally to be greater in DOG than DOPC (65 vs. 24%). Density functional theory calculations indicate that iron coordination by the carboxylate group of the propionate side chain, not the propionic acid group, is feasible in the aggregate. This can only occur, however, if ferrihaem is in a zwitterionic form with an axial water ligand, one propionic acid and one propionate side chain. UV-visible spectroscopy indicates that the presence of solvent water molecules is crucial for maintaining this species over a tautomeric form consisting of two propionic acid groups and an axial hydroxide ligand. Anhydrous conditions promote the latter tautomer which is unlikely to form Hz. A mechanism describing the initial stages of Hz formation is proposed based on these findings, which supports proposals that Hz crystal growth occurs in the lipid sub-phase.