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

ELife
Ruming ChenDavid Ron

Abstract

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex.

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Citations

Jun 24, 2015·Proceedings of the National Academy of Sciences of the United States of America·Margarito RojasThomas E Dever
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Oct 10, 2021·Nature Structural & Molecular Biology·Yahui YanDavid Ron

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Methods Mentioned

BETA
nucleotide exchange
transfection
flow cytometry
size-exclusion chromatography
X-Ray
size exclusion chromatography
protein folding
gel filtration
size
electrophoresis

Software Mentioned

DISOPRED
GraphPad
CCP4
Phaser
coot
XDS
phenix
MolProbity
refine
GraphPad Prism

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