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

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Neuroscience

Background:

• Serine/threonine-protein phosphatases with EF-hands (PPEFs) are crucial conserved proteins involved in cellular processes.
• Dysregulation of PPEFs is linked to significant human diseases, including cancer and neurodegenerative disorders.
• The precise molecular mechanisms governing PPEF activity, particularly their regulation by calcium, remain largely unelucidated.

Purpose of the Study:

• To elucidate the structural basis of Serine/threonine-protein phosphatase with EF-hands (PPEF) regulation by calmodulin (CaM) and calcium ions (Ca²⁺).
• To understand the auto-inhibitory mechanism and Ca²⁺-mediated activation of the Drosophila melanogaster retinal degeneration C (RDGC) enzyme.

Main Methods:

• Cryogenic-electron microscopy (cryo-EM) was employed to determine the high-resolution structures of the RDGC/CaM holoenzyme in different Ca²⁺ conditions.
• Structural analysis focused on the interactions between CaM, the EF-hand domains, and the extended-IQ motif of RDGC.

Main Results:

• In the absence of Ca²⁺, the extended-IQ motif, stabilized by CaM and the EF-hand tandem, auto-inhibits RDGC by blocking substrate access to catalytic sites.
• Upon Ca²⁺ binding, CaM and the EF-hand tandem induce significant conformational rearrangements in the extended-IQ motif, thereby activating the enzyme.
• This Ca²⁺-dependent activation mechanism mediated by dual sensors is conserved across species, including mammals.

Conclusions:

• The study provides unprecedented mechanistic insights into the Ca²⁺-dependent activation of PPEFs.
• Understanding this activation pathway opens avenues for developing targeted therapeutic strategies for diseases associated with PPEF dysfunction.
• The findings highlight the critical role of structural dynamics in enzyme regulation and disease pathogenesis.