ABSTRACT Protein-protein interactions are important targets for various drug discovery campaigns. One such promising and therapeutically pertinent protein-protein complex is Pf AMA1- Pf RON2 involved in malarial parasite invasion into human red blood cells. A thorough understanding of the interactions between these macromolecular binding partners is crucial for designing better therapeutics against this age-old disease. Although crystal structures of several Pf AMA1- Pf RON2 complexes are available, the mechanism of how domain II loop associates with Pf RON2 is not clear. The current work investigates how the domain II loop of Pf AMA1 exerts its effect on the alpha helix of the Pf RON2, thus influencing the overall kinetics of this intricate recognition phenomenon. To this end, we have computationally simulated the dynamics and free energetics of domain II loop closing processes and identified a set of key amino acid residues of Pf RON2 helix which are essential for binding. The subsequent evaluation of the binding affinity of Ala-substituted Pf RON2 peptide ligands by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) validates the relative importance of the residues in context. Together, the combination of computational and experimental investigation reveals that the domain II loop of Pf AMA1 is in fact responsible for arresting the Pf RON2 molecule from egress, K2027 and D2028 of Pf RON2 being the determinant residues for the capturing event. Our study provides a comprehensive understanding of the molecular recognition event between Pf AMA1 and Pf RON2, specifically in the post binding stage, which could potentially open up new avenues to drug discovery against malaria. TOC GRAPHIC

Computational and experimental studies of the breathing motion of a protein loop: implications in PfAMA1-PfRON2 late-stage binding event