Characterization of the interaction between AMA1 and the binding ligand RON2 in Plasmodium falciparum

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The Plasmodium falciparum antigen apical membrane antigen 1 (AMA1) is considered a leading malaria vaccine candidate and a potential target of anti-malarial drugs. The interaction between AMA1 and rhoptry neck protein 2 (RON2), both secreted from the invading merozoite, is indispensable for the formation of the moving junction, which is required for a successful parasite invasion. The crystal structure of AMA1 in complex with a RON2L peptide, which corresponds to the binding region in RON2, revealed a stepwise binding mechanism involving a major displacement of the domain II loop (DII loop) in AMA1. In this thesis experiments examining three aspects of the interaction between AMA1 and RON2 are described. Firstly, a phage-display approach was used to identify the AMA1- binding region in PfRON2 as a disulphide-bonded loop located between two predicted transmembrane regions in the C-terminal region of RON2. A peptide corresponding to this region competes with various AMA1-binding ligands and efficiently inhibits host cell invasion. In the second part of this study a 19F NMR approach was developed to detect the conformational change in the DII loop induced by ligand binding to AMA1. All four wildtype tryptophans and tryptophans inserted into the DII loop were replaced by 5- fluorotryptophan. An increase in flexibility of the DII loop caused by binding of the RON2L peptide was reflected in a marked sharpening of the resonance from the inserted 5-fluorotryptophan. A small AMA1-binding molecule isolated from a fragment-based library was shown to induce a similar conformational change. This approach will be valuable in identifying and characterizing therapeutically relevant inhibitors of the AMA1- RON2 interaction. In the last part of the study, mice were immunized with AMA1 in complex with bound ligands (peptides RON2L, R1 and R3) to test the hypothesis that AMA1-ligand complexes would divert antibody responses towards more conserved epitopes in AMA1. Little evidence was found to support this hypothesis but this approach to inducing a broadly protective antibody response to AMA1 should be examined in more detailed experiments.