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; 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 modelling 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.

Plasmepsin V (PM V) 是一种胃蛋白酶样天冬氨酸蛋白酶，对疟原虫恶性疟原虫的生长至关重要。先前的研究表明，PM V 是一种内质网驻留蛋白酶，可加工寄生于宿主细胞的寄生虫蛋白。在红细胞内的无性复制期间以及在有性阶段配子体的形成期间，酶的消耗或抑制是致命的。间日疟原虫 PM V 的结构已通过 X 射线晶体学进行了表征，揭示了一个典型的胃蛋白酶折叠，其间断有分泌天冬氨酸蛋白酶不常见的结构特征；然而，这种独特结构的功能尚不清楚。在这里，我们使用寄生虫遗传学通过尝试用各种突变酶挽救致命的 PM V 消耗来探测这些结构特征。我们在 PM V 基因中发现了一个不寻常的 nepenthesin 1 型插入物，它对寄生虫生长和 PM V 活性至关重要。nepenthesin 插入物的诱变表明，插入物在 PM V 功能中的作用需要其氨基酸序列和支撑其结构的两个二硫键之一。此外，与马尔可夫状态建模配对的分子动力学模拟表明，猪笼草素插入物的突变可能通过多种机制从变构上影响 PM V 催化。总之，这些数据为恶性疟原虫 PM V 蛋白酶的结构提供了进一步的见解。nepenthesin 插入物的诱变表明，插入物在 PM V 功能中的作用需要其氨基酸序列和支撑其结构的两个二硫键之一。此外，与马尔可夫状态建模配对的分子动力学模拟表明，猪笼草素插入物的突变可能通过多种机制从变构上影响 PM V 催化。总之，这些数据为恶性疟原虫 PM V 蛋白酶的结构提供了进一步的见解。nepenthesin 插入物的诱变表明，插入物在 PM V 功能中的作用需要其氨基酸序列和支撑其结构的两个二硫键之一。此外，与马尔可夫状态建模配对的分子动力学模拟表明，猪笼草素插入物的突变可能通过多种机制从变构上影响 PM V 催化。总之，这些数据为恶性疟原虫 PM V 蛋白酶的结构提供了进一步的见解。