How multisite phosphorylation impacts the conformations of intrinsically disordered proteins

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• Phosphorylation significantly impacts intrinsically disordered proteins (IDPs), modulating their structure, dynamics, and biological functions.
• A systematic understanding of how phosphorylation affects diverse IDPs is currently lacking.
• IDPs play crucial roles in various cellular processes, making their regulation by post-translational modifications a key area of research.

Purpose of the Study:

• To systematically investigate the effect of multi-site phosphorylation on the collapse propensity of four distinct intrinsically disordered proteins.
• To compare the accuracy of standard and tailored molecular dynamics force fields in predicting phosphorylation-induced changes in IDPs.
• To elucidate the influence of phosphorylation on protein solvation and active site exposure.

Main Methods:

• Utilized extensive all-atom molecular dynamics (MD) simulations to model unphosphorylated and phosphorylated forms of four IDPs: Ash1, CTD2', E-Cadherin cytosolic domain, and p130Cas fragment.
• Compared simulation results with experimental data, including Small-Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) spectroscopy.
• Investigated the impact of varying salt concentrations on simulation accuracy, particularly concerning electrostatic interactions and salt bridge stabilization.

Main Results:

• Observed a charge-dependent "V-shape" change in collapse propensity upon phosphorylation: neutral/negatively charged IDPs expanded, while positively charged IDPs contracted.
• Found that common and IDP-specific force fields overestimate phosphorylation-induced structural changes.
• Achieved quantitative agreement with experimental data for Ash1 and CTD2' only when attenuating electrostatic interactions (e.g., at 350 mM salt concentration).
• Demonstrated that phosphorylation significantly impacts protein solvation and can lead to substantial changes in active site exposure, even with mild global dimension changes.

Conclusions:

• Phosphorylation-induced structural changes in IDPs are strongly dependent on their net charge.
• Current molecular dynamics force fields require refinement, particularly in modeling electrostatic interactions, to accurately capture phosphorylation effects on IDPs.
• Beyond global dimensions, phosphorylation-induced alterations in solvation and active site accessibility are critical factors for understanding IDP regulation and function.