Abstract
Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction.
Highlights
Malaria is a deadly infectious disease caused by protozoan parasites of the genus Plasmodium
Using Surface plasmon resonance (SPR) and NMR spectroscopy we have validated that R1 binds to Apical membrane antigen 1 (AMA1) in solution with 1:1 stoichiometry, as suggested by previous ITC data [27], and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex
The hydrophobic segment Phe5-Leu6-Pro7-Leu8-Phe9, residues Phe12 and Arg15 are those that contribute most to the AMA1 binding affinity. They interact with two distinct binding hot spots, which are located at the two ends of the hydrophobic cleft of AMA1. Both of the pockets are highly conserved across the P. falciparum strains and likely to be suitable for designing broadspectrum AMA1 inhibitors
Summary
Malaria is a deadly infectious disease caused by protozoan parasites of the genus Plasmodium. In our current understanding of the structure and function of the MJ, AMA1 presents a conserved hydrophobic cleft that interacts with rhoptry neck protein 2 (RON2) [5]. This interaction is essential to the formation of the junction, which commits the parasite to invade [4,6]. Comparison with the structure of a complex between AMA1 and a peptide derived from RON2 reveals that the two peptides occupy the same region of AMA1 and exhibit structural mimicry [27] Consistent with these structural studies, R1 can effectively inhibit erythrocyte invasion by malaria parasites in vitro [25,28]. The inhibition is strain-specific, it has been demonstrated that N-methyl modification of R1 broadened its strain specificity [26]
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