Lande, E. S. | bioRxiv Science

bioRxiv2026

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.

biophysics↗

bioRxiv2026

Slow diffusion limits phosphorylation in a biomolecular condensate

Biomolecular condensates form dynamic compartments that regulate biochemical reactions in cells. Condensates recruit many kinases and regulate their enzymatic activity. Condensates alter the rate of enzymatic reactions through several opposing effects, so it is unclear whether these mostly enhance or retard phosphorylation. Here, we use a synthetic condensate formed by intrinsically disordered proteins to show that slow diffusion in the condensate controls phosphorylation kinetics in the dense phase. We vary the length of substrates by appending phase-separating repeat proteins of different lengths, in order to study how phosphorylation depends on partitioning, diffusion and volume fraction across substrate motifs with different intrinsic kinetics. The condensate environment is generally inhibitory to phosphorylation, although the enzyme remains intact. This inhibition is partially offset by an enhanced reaction rate in the dilute phase, likely due to soluble nanoclusters. Phosphorylation rates are strongly correlated to diffusion coefficients of substrates in the condensate, suggesting mass-transport limitation. Our results suggest that condensates can modify the substrate usage of a kinase via different trade-offs between diffusion and partitioning. We suggest that diffusion limitations are likely a common feature of many macromolecular reactions in condensates, and that high fluidity is crucial for condensates to act as reaction crucibles.

biophysics↗