Abstract
Plasmodium falciparum is an obligate intracellular protozoan parasite that employs a highly sophisticated mechanism to access the protective environment of the host cells. Key to this mechanism is the formation of an electron dense ring at the parasite-host cell interface called the Moving Junction (MJ) through which the parasite invades. The MJ incorporates two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 have shown that a partially mobile loop, termed the DII loop, forms part of a deep groove in domain I and overlaps with the RON2 binding site. To investigate the mechanism by which the DII loop influences RON2 binding, we measured the kinetics of association and dissociation and binding equilibria of a PfRON2sp1 peptide with both PfAMA1 and an engineered form of PfAMA1 where the flexible region of the DII loop was replaced by a short Gly-Ser linker (ΔDII-PfAMA1). The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants and corresponding thermodynamic profiles. Our results indicate that both PfAMA1 constructs bound to the PfRON2sp1 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. Consistent with Isothermal Titration Calorimetry measurements, final complex formation was enthalpically driven and slightly entropically unfavorable. Importantly, our experimental data shows that the DII loop lengthened the complex half-life time by 18-fold (900 s and 48 s at 25°C for Pf and ΔDII-Pf complex, respectively). The longer half-life of the Pf complex appeared to be driven by a slower dissociation process. These data highlight a new influential role for the DII loop in kinetically locking the functional binary complex to enable host cell invasion.
Highlights
Parasites in the phylum Apicomplexa include the etiological agents of malaria and toxoplasmosis
A ΔDII-PfAMA1 recombinant protein was constructed by replacing the segment KQYEQ HLTDYEKIKEGFKNKNASMIKSAFLPTGAFKA within the PfAMA1 DI-DII construct with 7 Gly/Ser residues
The truncated form ΔDII-PfAMA1 showed a 2-fold weaker affinity to the same PfRON2sp1 (92 nM). These results suggested an intriguing mechanism whereby the PfAMA1 DII loop enables a tighter interaction with PfRON2sp1
Summary
Parasites in the phylum Apicomplexa include the etiological agents of malaria and toxoplasmosis. Despite continuous efforts in vaccine development, prevention of malaria remains difficult and the spread of drug-resistant parasites highlights the critical need for new antimalarial strategies [2]. The MJ is initiated by injection of the Rhoptry Neck (RON) complex into the host cell, where RON2 spans the membrane and functions as a receptor for the Apical Membrane Antigen 1 (AMA1) protein located on the parasite surface [4,5,6,7]. Antibodies or peptides that prevent formation of the AMA1-RON2 complex block invasion [10,11,12,13,14,15,16,17]. Disrupting the AMA1-RON2 complex offers new strategies for the development of anti-infectives
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