| Popis: |
Plasmodium falciparum and Toxoplama gondii are obligate intracellular protozoan parasites that invade and replicate within host cells. They both require the formation of a tight interaction with the host cell, called Moving Junctions (MJ), for successful infection. It has been shown that the MJ contains 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 proteins have shown a versatile loop, called domain II loop that extends into domain I likely to hide the RON2 binding site from host immunity. In the present work, we have studied association, dissociation reactions and binding equilibria of PfAMA1 and TgAMA1 reacting with their respective RON2 short peptide ligand. Equally, we have studied a deltaDII-loop-PfAMA1 construct to elucidate the role of this loop upon RON2 peptide binding. The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants to calculate the thermodynamic profile and propose a reaction mechanism. Our results showed that PfAMA1 and TgAMA1 bind to their respective RON2 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. The reactions are both enthalpically and entropically favorable upon ligand binding thanks of the DII-loop induced fit folding down over the bound ligand forming a most stable final complex. The half life time of the complex at 25°C is 326s and 1077s for Pf and Tg complexes, respectively. By in vitro-in vivo extrapolation at 37°C, it is compatible with the time frame of erythrocyte invasion by Plasmodium falciparum merozoites. The elucidation of the binding mechanism brings new strategies for ligand discovery against these pharmacologically important targets. |
