The malaria antigen apical membrane antigen 1 (AMA1) is a protein of the Plasmodium parasite that is involved in the formation of the moving junction during red blood cell infection (1). The moving junction is formed by the interaction of rhoptry neck protein 2 (RON2) from the parasite with the hydrophobic cleft region on AMA1 (1). Invasion inhibitory molecules, R1 peptide and anti-AMA1 antibodies bind to the cleft and block the invasion process (2). Thus, AMA1 has become a target for drug development (2, 3). Here we describe the application of solution NMR spectroscopy to study AMA1-small molecule interactions. Based on perturbations in specific chemical shifts in the NMR spectrum of AMA1, it is feasible to map the binding sites for ligand/small inhibitory molecules, facilitating the design of new drugs to treat malaria.
The fragment of AMA1 we are studying is 42 kDa and this high molecular mass makes its chemical shift analysis particularly challenging because of resonance overlap. We have used different isotope labelling approaches including fractional deuteration, amino acid selective labelling and unlabelling to simplify and speed up the process of sequence-specific resonance assignments (4, 5). Partial backbone assignments were obtained from triple resonance NMR spectra and the spectra of selectively unlabelled AMA1 samples. AMA1-small molecule interaction studies were also carried out using 2D 15N TROSY experiments to map the binding sites for small molecules on AMA1. This information is being used to design high affinity ligands that block the binding of RON2 to the hydrophobic cleft of AMA1.