Structural dynamics of the wild-type p53 DNA-binding domain and hotspot mutants reveal oncogenic conformational shifts

The tumor suppressor protein p53, widely known for the potency and diversity of its functions, acts as a critical barrier to tumorigenesis. Mutations in p53, particularly within its DNA-binding domain (DBD), compromise its tumor suppressing function in over 40% of human tumors. Diverse p53 mutants adopt three major types of oncogenic effects, namely the loss-of-function effect, dominant-negative effect and gain-of-function effect. However, the conformational mechanisms by which hotspot mutations (<i>e.g.</i>, R175H, R273H/C) drive p53 dysfunction remain elusive. Here, we performed microsecond-level molecular dynamics simulations to dissect the structural dynamics of wild-type p53DBD and three oncogenic mutants. In wild-type p53DBD, multi-state conformational switching of the L1 loop was governed by hydrophobic interactions (A119/V122-P278) and an intra-loop hydrogen bond network. Notably, a previously unidentified β-hairpin conformation within the L1 loop was discovered, suggesting a latent regulatory motif. Mutations at R273 disrupted the H2 α-helix integrity, inducing helix-to-coil transitions that destabilized the DNA-binding interface. In contrast, R175H mutation triggered allosteric flexibility in both L2 and L3 loops, distorting the DNA contact surface through synergistic loop rearrangements. Interaction network analysis further revealed that these mutations remodeled non-local residue couplings, with R273H/C primarily destabilizing local interactions and R175H perturbing long-range communication with the LSH motif. Our findings provide structural insights into wild-type p53's complex activities and link mutation-specific conformational shifts to p53's loss/gain-of-function phenotypes, offering new avenues for restoring p53 activity in cancers.