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Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme

DOI: 10.7554/eLife.61509 DOI Help

Authors: Roman O. Fedoryshchak (Francis Crick Institute) , Magdalena Přechová (Francis Crick Institute) , Abbey Butler (Francis Crick Institute) , Rebecca Lee (Francis Crick Institute) , Nicola O'Reilly (Francis Crick Institute) , Helen R Flynn (Francis Crick Institute) , Ambrosius P Snijders (Francis Crick Institute) , Noreen Eder (Francis Crick Institute) , Sila Ultanir (Francis Crick Institute) , Stephane Mouilleron (Francis Crick Institute) , Richard Treisman (Francis Crick Institute)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Elife , VOL 9

State: Published (Approved)
Published: September 2020
Diamond Proposal Number(s): 9826 , 9826 , 18566

Open Access Open Access

Abstract: PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally-enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin aII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.

Journal Keywords: Phactr1; Protein Phosphatase 1; PP1; RPEL; Actin; Cytoskeleton Dephosphorylation; IRSp53; Spectrin; Afadin

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Added On: 30/09/2020 09:33

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)