Plasmepsin V (PM V) is a pepsin-like aspartic protease essential for growth of the malarial parasite Plasmodium falciparum. Previous work has shown PM V to be an endoplasmic reticulum-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 Plasmodium vivax PM V has been characterized by X-ray crystallography, revealing a canonical pepsin fold punctuated by structural features uncommon to secretory aspartic proteases; however, the function of this unique structure is unclear. Here, we used parasite genetics to probe these structural features by attempting to rescue lethal PM V depletion with various mutant enzymes. We found an unusual nepenthesin 1-type insert in the PM V gene 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 insert's role in PM V function. Furthermore, molecular dynamics simulations paired with Markov state modeling suggest that mutations to the nepenthesin insert may allosterically affect PM V catalysis through multiple mechanisms. Taken together, these data provide further insights into the structure of the P. falciparum PM V protease.

01 Apr 2022·Protein ScienceQ4 · BIOLOGY

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

Q4 · BIOLOGY

Article

Author: Deshmukh, Anuradha ; Suryawanshi, Abhishek B. ; Wlodawer, Alexander ; Edwards, Thomas E. ; Pahelkar, Nikhil ; Yada, Rickey Y. ; Lorimer, Donald D. ; Gustchina, Alla ; Xiao, Huogen ; Mishra, Vandana ; Labaied, Mehdi ; Kesari, Pooja ; Bhaumik, Prasenjit ; Abendroth, Jan ; Dupuis, John H. ; Rathore, Ishan

AbstractPlasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin‐like aspartic proteases from apicomplexan parasites.

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