In particular, this composite pocket is located next to the part of the PP1 enzyme responsible for phosphate removal. This showed that binding of Phactr1 to PP1 creates a new surface pocket, which comprised elements of both proteins. First, the structure of the Phactr1/PP1 complex was examined using biochemistry approaches and X-ray crystallography. studied a phosphatase called PP1 and its partner, Phactr1. To investigate this, Fedoryshchak, Přechová et al. Partner proteins can attach to phosphatases to bring the enzymes to specific locations in the cell, or to deliver target proteins to them yet, it is unclear whether partner binding could also change the structure of the enzyme so the phosphatase can recognise only a restricted set of targets. Phosphatases, however, are generalists: only a few different types exist, which exhibit little target sequence specificity. Kinases are generally specific for a small number of proteins, adding phosphate groups only at sites embedded in a particular sequence in the target protein. Enzymes called kinases are responsible for adding these groups onto a protein, while phosphatases remove them.
Specific arrangements of atoms such as bulky phosphate groups can change the activity of a protein and how it interacts with other molecules.
Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism.
We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. PPP-family phosphatases such as PP1 have little intrinsic specificity.
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