Structural insights into the dual Ca2+-sensor-mediated activation of the PPEF phosphatase family

Serine/threonine-protein phosphatases with EF-hands (PPEFs) are a family of highly conserved proteins implicated in cancer and neuronal degeneration. The initially characterized member, Drosophila melanogaster retinal degeneration C (RDGC) contains a calmodulin (CaM)-interacting extended-IQ motif and a Ca²⁺-binding EF-like/EF-hand tandem. However, the molecular regulation of PPEF is poorly understood. In this study, we use cryogenic-electron microscopy to delineate the structures of the RDGC/CaM holoenzyme. In the absence of Ca²⁺, CaM and the EF-like/EF-hand tandem allow the extended-IQ motif to block substrate access to the catalytic sites, constituting an auto-inhibitory mechanism. Upon Ca²⁺ binding, CaM and the EF-like/EF-hand tandem drive drastic conformational changes in the extended-IQ motif to unlock the catalytic sites. This dual Ca²⁺-sensor-mediated activation is evolutionarily conserved in mammals. This study provides mechanistic insight into the molecular activation of PPEFs, paving the way for the development of therapeutic strategies for PPEF-related human diseases.