G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

Area of Science:

• Molecular Biology
• Biochemistry
• Cellular Stress Response

Background:

• Protein synthesis recovery after cellular stress relies on dephosphorylating eukaryotic translation initiation factor 2a (eIF2a).
• This process is mediated by protein phosphatase 1 (PP1) in conjunction with regulatory subunits PPP1R15A (GADD34) or PPP1R15B (CReP).
• Binary PPP1R15-PP1 complexes lack substrate selectivity in vitro.

Purpose of the Study:

• To investigate the mechanism by which PPP1R15-PP1 complexes achieve substrate selectivity.
• To elucidate the role of G-actin in modulating the activity and specificity of these dephosphorylation complexes.

Main Methods:

• Reconstitution of PPP1R15-PP1 binary complexes in vitro.
• Formation and structural analysis of a ternary complex including PPP1R15, PP1, and G-actin.
• X-ray crystallography to determine complex structures.
• Computational docking to model substrate interaction.
• Site-directed mutagenesis to assess residue importance.

Main Results:

• G-actin binding to PPP1R15-PP1 complexes restored substrate selectivity.
• Crystal structures revealed actin's role in forming an active site platform in the selective ternary complex.
• Computational docking and mutagenesis data suggest substrate avidity is key for specificity.

Conclusions:

• The PPP1R15B-PP1G-actin ternary complex exhibits substrate selectivity, unlike binary complexes.
• Actin binding is essential for creating a specific substrate-binding platform.
• Substrate avidity plays a critical role in conferring specificity to the eIF2a dephosphorylation machinery.