Affinity and distance dependence of SLiM-mediated dephosphorylation by protein phosphatase 1

Protein phosphatases counterbalance kinases by dephosphorylating phospho-proteins to regulate signaling pathways. But unlike kinases, their shallow catalytic groove has limited selectivity for the phospho-peptide motifs. Phosphatases such as protein phosphatase-1 (PP1) recognize their substrates through short linear motifs (SLiMs) within intrinsically disordered regions, yet the mechanism of SLiM-mediated recruitment remains unclear. We developed an intrinsically disordered phospho-protein substrate to enable quantitative modelling of tethered PP1 catalysis by varying SLiM affinity and distance. We show that a PP1-binding SLiM linked to a phospho-peptide by a flexible spacer is sufficient to enhance dephosphorylation. Dephosphorylation occurs most efficiently at a spacing of 20-30 residues, in agreement with predictions from theoretical polymer models. Low micromolar dissociation constants are most efficient for dephosphorylation, which can be modelled numerically as a trade-off between substrate binding and substrate inhibition. Bivalent SLiMs enhance or decrease catalysis depending on the combined affinity. Together, these results define a quantitative and generalizable framework for how linear motifs direct dephosphorylation by PP1.