T253X Mutations Alter Local Structure and Trigger Tyr236 Hydration in p53 DNA-Binding Domain

Mutations in the DNA-binding domain (DBD) of the p53 tumor suppressor are common in human cancers and frequently compromise its structural integrity or DNA-binding activity. Threonine 253 (Thr253) lies within the S9 aggregation-prone region, and multiple clinically reported substitutions at this position have been observed in patients with inherited cancer predisposition. However, the structural consequences of these mutations remain unclear. Here, we investigate the conformational dynamics of the wild-type (WT) p53 DBD and four T253 variants (T253A, T253P, T253I, and T253N) using all-atom molecular dynamics simulations. These simulations show that the T253 mutations remodel a local stability element centered on Tyr236, residue 253, and the S6-S7/S9-S10 loop ensemble. All T253 variants abolish or weaken the Tyr236-Thr253 hydrogen bond in the hydrophobic core. This disruption converts Tyr236 from a dry residue in WT to one that forms hydrogen bonds with water molecules, representing a common local response to mutations at position 253. Our results identify the Tyr236-residue 253 region as a local stability hotspot where buried polarity and steric bracing regulate access to an underlying β-sheet. They further provide a mechanistic basis for interpreting nonhotspot T253 variants that preserve the global fold yet subtly rewire buried interactions and hydration in a functionally critical segment.