Two Distinct Domains Mediate Self-Assembly of Knob Protein in Plasmodium Falciparum-Infected Human Erythrocytes.

Plasmodium falciparum associated mortality is believed to originate from the adhesion of infected erythrocytes to the endothelial cells. The adhesion of mature parasite infected erythrocytes is mediated by the surface protrusions called knobs. A dominant constituent of knobs is the parasite derived knob-associated histidine-rich protein termed KAHRP or knob protein. Gene deletion experiments have shown that the knob protein is essential in malaria pathogenesis. Without the knob protein (KAHRP), adherence of malaria-infected erythrocytes is not established under physiological shear stress. The adhesive properties of infected erythrocytes are propagated by the direct interaction of KAHRP, a peripheral membrane protein, with the cytoplasmic domain of the P. falciparum erythrocyte membrane protein-1 (PfEMP1). Unlike the highly polymorphic extracellular domain, the cytoplasmic domain of PfEMP1 is conserved across species, hence the interest in delineating the mechanism of its interaction with KAHRP and components of the erythrocyte membrane. Our studies have observed recombinant KAHRP to self-associate into large aggregates in vitro. Under transmission electron microscopy, both knobs isolated from malaria-infected erythrocytes and self-assembled recombinant KAHRP in vitro have revealed a unique spiral shaped morphology. Based on these observations, we hypothesized that the unique knob spiral is formed by the self-assembly of KAHRP monomers and therefore must be encoded by distinct sequence elements within KAHRP. To investigate this speculation, we devised a solution binding assay and engineered a series of GST (glutathione S-transferase) and Trx (thioredoxin) fusion peptides to encompass the entire length of KAHRP. Binding measurements using heterologous combination of defined fusion peptides reveal a core histidine-rich amino terminal segment associating with a lysine-rich segment located within the carboxyl half of KAHRP. These findings suggest a novel head-to-tail interaction strategy employed by the intracellular parasite to assemble the spiral-shaped scaffold of KAHRP under the surface of electron dense knobs. Significantly, our results identify a novel self-interaction site within KAHRP that could be amenable to therapeutic intervention against cerebral malaria.