Apical membrane antigen 1 (AMA1) protein is an essential component for invasion of host cells by zoites of all apicomplexan parasites. Another key player, rhoptry neck protein 2 (RON2), is inserted into the host cell membrane during invasion and acts as a ligand for AMA1. Several studies indicate that the AMA1/RON2 interaction is essential to malaria invasion. 1,2 A range of small molecule ligands for AMA1 has been identified in a fragment screen and shown to bind to the conserved hydrophobic cleft of PfAMA1. However, crystal structures of AMA1 with these ligands bound failed to identify their binding site(s). 3 We have therefore pursued alternative approaches based on NMR to define their binding sites.
A spin-labelled peptide was developed to probe the binding sites of compounds on PfAMA1. A thio-specific stable nitroxide spin label was attached to a solvent-exposed cysteine of a backbone-cyclised peptide derived from PfRON2. Its Kd was 1 µM by SPR. A 1.6 Å resolution crystal structure of the complex shows that the peptide binds to PfAMA1 FVO at one end of the hydrophobic groove and leaves much of the binding site exposed, allowing elaborated fragments to bind without interference.
Paramagnetic relaxation enhancement (PRE)-based NMR experiments were carried out to observe the peptide’s effects on AMA1. The relaxation rates of 1H nuclei near the spin are enhanced owing to distance-dependent paramagnetic effects that can be detected as line broadening or reduced NMR peak intensities. Thus, distance constraints can be inferred in order to identify the binding sites of elaborated fragments and serve to guide for antimalarial drug design. The spin label has local PRE effects on residues around the cleft as well as some long-range effects that could arise from non-specific binding of the peptide.