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Towards In Situ Dynamics of DNA-bound Full-Length p53 Tetramer
Ozlem Demir1,2, Emilia P Barros1,3, Rommie E Amaro4
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.
Abstract:
p53 is the most important tumor suppressor in humans as well as the most frequently mutated gene found in human cancers with ~50% of all human tumors bearing p53 missense mutations that leave p53 inactive. Restoring the p53 activity proved to lead to tumor regression even in advanced tumors in mouse models- and thus, is among the most attractive potential strategies for novel cancer therapy. Full-length p53 (fl-p53) consists of 393 residues and multiple domains; some folded and some disordered. Using crystal structures of folded domains and integrative molecular modelling techniques for disordered domains, we generated the first wild-type fl-p53 tetramer model bound to DNA. When solvated, the system size nears 500K atoms challenging extensive sampling. Using Anton2 supercomputer for microsecond-timescale simulations in explicit solvent and the rigorous Markov state model (MSM) framework, we elucidated the conformational landscape of wild-type p53 as well as two of the p53 hot-spot cancer mutants, Y220C and G245S, in a physiological DNA-bound, full-length tetramer context. In the simulated timescale, DNA-bound fl-p53 tetramer bent DNA and formed a compact complex with interactions between the N-terminal and DNA-binding domains (DBDs), and the C-terminal domains (CTDs) with DNA. WT fl-p53 tetramer also sampled a unique quaternary DBD organization not accessed by the cancer mutants. Free energy landscapes indicated differential dynamics for inner and outer p53 DBDs due to the dimer-dimer interface. The dynamics of the druggable L1/S3 pocket is also closely monitored. Ultimately the MSMs identified an underexplored loop 6 (L6) cryptic pocket and captured the effect of p53 tetramerization and cancer mutations.
Insights
Restoring tumor suppressor p53 activity is a promising cancer therapy. This study models the full-length p53 tetramer, revealing its DNA interactions and dynamics, including cancer mutations.
Area of Science:
- Molecular Biology
- Biophysics
- Computational Biology
Background:
- The p53 protein is a critical human tumor suppressor, frequently inactivated by mutations in ~50% of cancers.
- Restoring p53 activity is a highly sought-after cancer therapy strategy, showing promise in preclinical models.
- Full-length p53 (fl-p53) is a multi-domain protein whose structure and dynamics in a physiological context are complex.
Purpose of the Study:
- To generate the first atomic model of the wild-type full-length p53 tetramer bound to DNA.
- To investigate the conformational landscape and dynamics of wild-type and mutant p53 using advanced computational simulations.
- To understand how p53 tetramerization and cancer mutations affect its DNA-binding and overall structure.
Main Methods:
- Integrative molecular modeling combining crystal structures and disordered domain modeling.
- Microsecond-timescale molecular dynamics simulations on the Anton2 supercomputer in explicit solvent.
- Markov state modeling (MSM) to analyze the conformational landscape and identify key dynamic features.
- Analysis of DNA bending, protein-protein interactions, and druggable pocket dynamics.
Main Results:
- A detailed model of the DNA-bound wild-type fl-p53 tetramer was generated, revealing DNA bending and compact complex formation.
- Differential dynamics were observed between inner and outer p53 DNA-binding domains (DBDs) due to the dimer-dimer interface.
- Wild-type p53 tetramer adopted a unique quaternary DBD organization not seen in the analyzed cancer mutants (Y220C, G245S).
- An underexplored cryptic pocket in loop 6 (L6) was identified, and the impact of tetramerization and mutations on its dynamics was captured.
Conclusions:
- The study provides unprecedented insights into the structural dynamics of the full-length p53 tetramer bound to DNA.
- Cancer mutations and tetramerization significantly alter p53's conformational landscape and dynamics, particularly affecting DBD organization and cryptic pocket accessibility.
- These findings lay the groundwork for developing novel therapeutic strategies targeting p53 reactivation in cancer.
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