Activation mechanism of plasmepsins, pepsin‐like aspartic proteases from Plasmodium, follows a unique trans‐activation pathway

Abstract

Plasmodium parasites that cause malaria produce plasmepsins (PMs), pepsin‐like aspartic proteases that are important antimalarial drug targets due to their role in host hemoglobin degradation. The enzymes are synthesized as inactive zymogens (pro‐PMs), and the mechanism of their conversion to the active, mature forms has not been clearly elucidated. Our structural investigations of vacuolar pro‐PMs with truncated prosegment (pro‐tPMs) reveal that the formation of the S‐shaped dimer is their innate property. Further structural studies, biochemical analysis, and molecular dynamics simulations indicate that disruption of the Tyr‐Asp loop (121p‐4), coordinated with the movement of the loop L1 (237–247) and helix H2 (101p–113p), is responsible for the extension of the pro‐mature region (harboring the cleavage site). Consequently, under acidic pH conditions, these structural changes result in the dissociation of the dimers to monomers and the protonation of the residues in the prosegment prompts its unfolding. Subsequently, we demonstrated that the active site of the monomeric pro‐tPMs with the unfolded prosegment is accessible for peptide substrate binding; in contrast, the active site is blocked in folded prosegment form of pro‐tPMs. Thus, we propose a novel mechanism of auto‐activation of vacuolar pro‐tPMs that under acidic conditions can form a catalytically competent active site. One monomer cleaves the prosegment of the other one through a trans‐activation process, resulting in formation of mature enzyme. As a result, once a mature enzyme is generated, it leads to the complete conversion of all the inactive pro‐tPMs to their mature form.DatabaseAtomic coordinates and structure factors have been submitted in the Protein Data Bank (PDB) under the PDB IDs 6KUB, 6KUC, and 6KUD.

Structures of plasmepsin X from P. falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme

“…Analysis of the arrangement of the protein molecules in the crystals reveals that Pfpro-PMX forms an "S" shaped dimer, also observed in zymogens of other vacuolar PMs 22 . The loop region of the prosegment consisting of residues N225p-K232p is swapped between the two adjacent monomers of the dimer (Extended Data Figs.…”

“…Pfpro-PMX has been compared with the representative zymogen structures of other pepsin-like aspartic proteases from parasites, plants, and animals. Previously reported crystal structures of the truncated zymogens of P. falciparum vacuolar plasmepsins (PfPMII, PfHAP, and PfPMIV) reveal that their prosegments have an initial β-strand, followed by two α-helices connected by a turn and a coiled extension with the characteristic "Tyr-Asp" loop that connects to the mature segment of the enzyme 22 . The prosegment of the plant and gastric pepsin-like aspartic protease zymogen comprises the first β-strand, followed by two α-helices, a short α-helix, and a turn connecting to the mature domain of the enzyme.…”

“…The α1 of Pfpro-PMX points downwards, occupying the same space as the tip of a loop in PfPMII zymogen (115p-126p) and pepsinogen; hence similar loop is absent in the Pfpro-PMX structure. This loop of vacuolar PM zymogens plays an important role in their activation mechanism 22 ; the absence of such structural element in Pfpro-PMX may imply a different mechanism of maturation. The next secondary structural elements in pepsinogen and PfPMII zymogen are α-helices pointing in two different directions; in Pfpro-PMX it is a β-strand.…”

“…The last part of the prosegment in pepsinogen forms a short helical turn that provides a lysine residue blocking the active site aspartates; an equivalent position is partly occupied by the twisted loop region of Pfpro-PMX. In vacuolar PM zymogens the second α-helix is involved in formation of the inter-dimer contacts and the "Tyr-Asp" loop keeps the prosegment in a strained conformation 22 . Inactivation of vacuolar PM zymogens is achieved by the prosegment that acts as a harness separating the two domains of the enzyme, resulting in distancing of the catalytic aspartates.…”

“…In general, activation of the zymogens of pepsin-like aspartic proteases takes place by removal of the prosegment under acidic conditions. It is suggested that vacuolar PMs have an auto-activation mechanism wherein the dimeric form is converted to a monomer under acidic conditions due to the disruption of the Tyr-Asp loop at the pro-mature junction, with subsequent unfolding and cleavage of the prosegment by the trans-activation process 22 . Conversely, in other pepsin-like aspartic proteases, the interaction of a lysine residue with the catalytic aspartates is responsible for inactivation of the zymogen, which then detaches from the active site under acidic pH, leading to the unfolding of prosegment followed by cleavage by the same enzyme molecule 20,23 .…”