Understanding the Activation Mechanism of Proplasmepsins and the Catalytic Mechanism of Aspartic Proteases With Implications in Drug Development

Plasmodium parasites that cause malaria produce plasmepsins (PMs), the pepsin-like aspartic proteases. These enzymes, synthesized as inactive zymogens (proPMs), are important antimalarial drug targets due to their role in host hemoglobin degradation. The mechanism of conversion of proPM to its active mature form has not been clearly elucidated. Our structural investigations reveal that the formation of the S-shaped dimer is an innate property of proPMs. The structural studies, biochemical analysis, and molecular dynamics simulations suggest that disruption of the Tyr-Asp loop (121p-4), coordinated with the movement of the loop (L1; 237-245) and helix (H2; 101p-113p) are responsible for the extension of the pro-mature region (harboring the cleavage site), leading to the dissociation of a dimer to monomers. In the acidic pH, the pro-mature region of the monomer adopts a more extended conformation, and protonation of the residues in the prosegment prompts it’s unfolding. We further observed that the active site of the zymogen with the unfolded prosegment is accessible for peptide binding, in contrast to the folded form wherein the active site is blocked. We propose a novel mechanism of auto-activation of vacuolar PM zymogens, which under acidic conditions form a catalytically competent active site and one subunit cleaves the prosegment of the other through a trans-cleavage process, resulting in formation of the first molecule of mature enzyme. The most potent of inhibitors against an enzyme mimic the transition state of the reaction. The success of these kind of inhibitors hugely depend on the correctness of the known mechanism. Mechanism based inhibitors has greater scope of success due to tighter binding and are less likely to be overcome by point mutations. Thus, a better understanding of the catalytic mechanism is warranted in designing potent mechanism based inhibitors against aspartic proteases. Although aspartic proteases have been studied for decades, some of the finer details of the mechanism are not clearly understood. In our study to decipher the binding of the peptide and the position of the water molecule in the initial step of the reaction, we have employed the use of Plasmepsin II (PMII) as a model system and peptidomimetic KNI inhibitors. Here we have solved three crystal structures of PMII bound with KNI inhibitors (10006, 10772 and 10773). Interestingly, we have found that modifications in the functional groups of these inhibitors alter their binding mode. Moreover, in the crystal structure of KNI-10773 bound PMII, we have captured the catalytic water molecule, possibly when the peptide is bound in the active site. In our inhibitor bound structures, the active site aspartates are found to be non-planar which is unusual and contradictory to earlier reported studies. Based on the crystal structures and structural studies, we propose important modification to the current mechanism of pepsin-like aspartic proteases, where the non-planar conformation of Asp216 is responsible for generation of the nucleophile. Thus, our study provides a significant improvement in the understanding of both the activation and the catalytic mechanism of aspartic proteases will help in designing more potent mechanism based inhibitors.