A nepenthesin insert allosterically controls catalysis in the malaria parasite protease plasmepsin V

Plasmepsin V (PM V) is a pepsin-like aspartic protease essential for growth of the malaria parasite Plasmodium falciparum . Previous work has shown PM V to be an ER-resident protease that processes parasite proteins destined for export into the host cell. Depletion or inhibition of the enzyme is lethal during asexual replication within red blood cells, as well as during the formation of sexual stage gametocytes. The structure of the P. vivax PM V has been characterized by x-ray crystallography, revealing a canonical pepsin fold punctuated by structural features uncommon to secretory aspartic proteases. Here we use parasite genetics to probe these structural features by attempting to rescue lethal PM V depletion with various mutant enzymes. We find an unusual nepenthesin 1-type insert to be essential for parasite growth and PM V activity. Mutagenesis of the nepenthesin insert suggests that both its amino acid sequence and one of the two disulfide bonds that undergird its structure are required for the nepenthesin insert’s role in PM V function. Molecular dynamics simulations paired with Markov state modelling suggest that the nepenthesin insert allosterically controls PM V catalysis through multiple mechanisms.