Towards In Situ Dynamics of DNA-bound Full-Length p53 Tetramer

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. Significance p53, the most important tumor suppressor in humans, is found inactivated due to single point mutations in almost 50% of all human tumors. As restoring p53 activity is shown to achieve tumor regression even in advanced tumors in mice, there is a lot of interest in finding small molecules to reactivate p53 cancer mutants. p53 has 393 residues and is an intrinsically disordered protein with multiple unstructured and dynamic domains that are known to interact with and help the function of its folded domains. Adding another level of complexity, p53 forms a tetramer to bind DNA and start its transcriptional activity. Due to its high dynamicity, p53 evades full structural characterization leaving many open questions. We generated an integrative model weaving together structures of isolated p53 domains and explored it with molecular dynamics simulations at microsecond timescale. This allowed us to monitor various unresolved aspects with atomistic detail like the dynamics of druggable pockets, the interplay of p53 domains in the physiological complex and how they change in p53 cancer mutants compared to the wild-type p53. Our study provides insights into the structure and dynamics of a key tumor suppressor protein at its physiologically relevant state of full-length tetramer bound to DNA. Competing Interest Statement O. Demir and E. Barros are current employees of Novartis Biomedical Research and MSD, respectively, and own stocks and/or stock options in those companies.