Site-specific phosphorylation modulates p16/CDK4 binding dynamics and energetics: Insights from molecular simulations

Area of Science:

• Molecular Biology
• Structural Biology
• Computational Biophysics

Background:

• The tumor suppressor p16INK4a regulates the cell cycle by inhibiting cyclin-dependent kinase 4 (CDK4).
• Phosphorylation is a key post-translational modification that alters protein function, but its specific effects on p16INK4a's interaction with CDK4 are not fully understood.

Purpose of the Study:

• To investigate the structural and energetic consequences of site-specific phosphorylation on the p16INK4a/CDK4 complex.
• To decipher how phosphorylation influences the conformational dynamics, interfacial stability, and binding energetics of the p16INK4a/CDK4 interaction.

Main Methods:

• All-atom molecular dynamics simulations were used to study wild-type and phosphorylated p16INK4a variants in complex with CDK4.
• Analyses included regional flexibility, interfacial packing, binding free energy calculations, free energy landscapes, and surface electrostatics.

Main Results:

• Phosphorylation at specific sites (residues 8, 12, 93) led to rapid dissociation of the p16INK4a/CDK4 complex.
• Other phosphorylation sites resulted in weakened interfacial packing and destabilization.
• Terminal phosphorylation (S152Sp, S56Sp, S7Sp) showed potential stabilizing effects, retaining wild-type binding modes.

Conclusions:

• Phosphorylation dynamically modulates p16INK4a's interaction with CDK4, with site-specific outcomes ranging from destabilization to stabilization.
• These findings provide mechanistic insights into cell cycle control by p16INK4a and offer a molecular basis for understanding cancer-related dysregulation of the p16INK4a/CDK4 pathway.