Structural techniques, such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, require multi-milligram quantities of material. Studying how phosphorylation impacts protein structure and protein-protein interactions is a fundamentally important yet material-intensive endeavor. 1 The folded SAP domain is necessary for a high-affinity and reversible interaction with the MUS81-EME1 endonuclease in early mitosis. Recently, we showed that phosphorylation of the MUS81-binding region in SLX4 (SLX4 MBR) triggers folding of a canonical SAP domain. 12 As such, phosphorylation of 4E-BP2 negatively regulates its interaction with eIF4E. However, multi-site phosphorylation of 4E-BP2 induces the folding of a four-stranded β-domain that sequesters the helical motif that is required for the interaction with eIF4E. 12, 13, 14 Binding of eIF4E drives a disorder-to-helix transition in 4E-BP2. 11 A well-characterized example of phosphorylation-induced protein folding is the intrinsically disordered protein 4E-BP2 and its interaction with eIF4E. 10 Phosphorylation-induced unfolding has also been observed in the case of the ankyrin-repeat protein p19(INK4d), which functions as a key regulator of the G1/S cell cycle transition. For example, phosphorylation drives partial unfolding of the C-terminus of the Hsp90 co-chaperone Cdc37. Phosphorylation can also exert changes in protein folding with important functional implications. 8 Despite the widespread occurrence of phosphorylation, less than 3% of the identified sites in the human phosphoproteome have been linked to a specific function. Although the effects of phosphorylation on protein-protein interactions can be diverse, one common theme is the regulation of binding affinity. Some of these effects result from phosphorylation-driven changes in protein-ligand interactions. 3, 4, 6, 7 Phosphorylation can also regulate protein sub-cellular localization, stability, and activity. Protein phosphorylation acts as a reversible switch in signaling pathways that regulate nearly all aspects of cellular homeostasis, including growth and proliferation, DNA repair, and cell division. Notably, phosphorylation is frequently observed on residues that occupy disordered regions with low sequence conservation. An important cofactor in this reaction is magnesium, which chelates the γ- and β-phosphate groups and is crucial for phosphoryl transfer to the substrate. 2, 3 The hydroxyl side-chain provides for the nucleophilic attack of the terminal phosphate (i.e., γ-phosphate) in adenosine 5′-triphosphate (ATP), driving the transfer of the phosphoryl group to the protein substrate. 4 Mechanistically, kinases catalyze the addition of a phosphoryl group to specific amino acid residues in the target protein, predominately serine, threonine, and tyrosine in eukaryotes. 2, 3 This PTM is regulated by the opposing actions of kinases and phosphatases, which catalyze the phosphorylation and dephosphorylation of target proteins, respectively. Protein phosphorylation is one of the most common post-translational modifications (PTMs) in eukaryotes, owing in part to its reversible nature.
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