Higher-order phosphatase-substrate contacts terminate the integrated stress response

Area of Science:

• Molecular Biology
• Structural Biology
• Cellular Signaling

Background:

• Protein phosphatase 1 (PP1c) catalytic subunits associate with regulatory PPP1R subunits to form holophosphatases.
• Regulatory subunits modulate PP1c activity by interacting with its surface, influencing substrate access.
• The precise mechanism of catalytic efficiency in holophosphatases remains poorly understood.

Purpose of the Study:

• To elucidate the structural basis of holophosphatase activity in the integrated stress response (ISR).
• To determine the structure of the PP1c-PPP1R15A-G-actin holophosphatase complex with its substrate, eIF2α.
• To understand the role of G-actin in substrate recognition and dephosphorylation.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of the holophosphatase-substrate complex.
• Crystallographic, biochemical, and genetic analyses to validate the role of G-actin.

Main Results:

• A cryo-EM structure of the tripartite PP1c-PPP1R15A-G-actin holophosphatase in complex with eIF2α was determined.
• G-actin acts as a scaffold, aligning PP1c and PPP1R15A to create a composite surface for eIF2α binding.
• This alignment precisely positions the phosphoserine-51 residue of eIF2α within the PP1c active site for dephosphorylation.

Conclusions:

• G-actin plays a critical structural role in enabling eIF2α dephosphorylation by the PP1c holophosphatase.
• Substrate residues mediating affinity for the holophosphatase also interact with eIF2α kinases, linking phosphorylation and dephosphorylation.
• Higher-order substrate recognition by the holophosphatase dictates functionally antagonistic phosphorylation and dephosphorylation events in the ISR.