Abstract
The development of novel antimalarial drugs requires the identification of parasite-specific vulnerabilities. Translation control, regulated by eIF2α dephosphorylation, is essential for Plasmodium development during infection. The parasite phosphatase UIS2 regulates this modification. However, the structural basis for UIS2 substrate recognition remains unknown. Here, we show that UIS2 is crucial for parasite development in both liver and blood stages. Its N-terminal and phosphatase domains each independently bind the eIF2α phosphoserine-59 loop through electrostatic interactions. Integrated structural modeling, using AlphaFold and molecular dynamics simulations, reveals a defined binding pocket within the phosphatase domain, with a geometry distinct from human PP1α. This pocket contains six cooperative binding patches that use an electrostatic network to stabilize the phosphopeptide near the active site and contribute to substrate specificity. Metadynamics simulations show that the inhibitor Salubrinal competes with the phosphopeptide for binding to this pocket. Molecular docking and free energy perturbation analyses show that Salubrinal targets three of these binding patches through hydrophobic and hydrogen-bonding interactions, blocking substrate binding. These findings show the structural and energetic basis of eIF2α dephosphorylation by UIS2. They provide an integrated modeling approach for phosphatase-substrate interactions and a mechanistic rationale for selective inhibition of a parasite-specific translation regulator.