Prodomain-driven enzyme dimerization, a potent pH-dependent autoinhibition mechanism to control Plasmodium Sub1 subtilisin-like activity prior to prime merozoite egress

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBC) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes (RBC). Over the last two decades it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi- step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto- maturation in the Endoplasmic Reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergo further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the parasitophorous vacuole (PV). Here we report the crystal structure of full- length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical and biophysical studies, that the atypical Plasmodium- specific Sub1 PD directly triggers the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, particularly the different compartmentalization of PmX processing and full Sub1 activation, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition involved in the tight regulation of P. falciparum merozoites egress from host infected cells. Significance Malaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin- like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is requested in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here we show that the unusual, parasite specific Sub1 prodomain is directly responsible for its acidic- dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.