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

Phosphorylation of the tumor suppressor p16INK4a is known to influence its inhibitory interaction with cyclin dependent kinase 4 (CDK4), yet the structural and energetic consequences of site specific modifications remain unclear. Here, we employed all-atom molecular dynamics simulations to systematically investigate Wild-type and single-site phosphorylated p16 variants in complex with CDK4, aiming to decipher phosphorylation driven changes in conformational dynamics, interfacial stability, and binding energetics. Region wise analyses revealed distinct alterations in flexibility and stabilization across the N-terminal, intermediate, and C-terminal segments of p16 upon phosphorylation. Notably, phosphorylation at residues 8, 12, and 93 triggered rapid dissociation of the p16/CDK4 complex, highlighting their critical role in maintaining interfacial integrity. Most other phosphorylated complexes exhibited weakened interfacial packing, suggesting a propensity toward destabilization. Binding free energy calculations indicated that van der Waals interaction is the main stabilizing force whereas, electrostatic contributions vary among different systems. Phosphorylated variants S152Sp, S56Sp and S7Sp retained binding modes similar to the Wild-type, supported by free energy landscapes and surface electrostatics, demonstrating a potential stabilizing role of terminal p16 phosphorylation. This work provides mechanistic insights into phosphorylation mediated tuning of p16 function in controlling the cell cycle and offers a molecular basis for understanding cancer associated dysregulation of the p16/CDK4, guiding future experimental validation and therapeutic exploration.