Loh, S. N. | bioRxiv Science

bioRxiv2021

Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D

Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that full-length p53 (FLp53) binds to CypD with KD of ~1 M, while both NTD-DBD and NTD bind to CypD at ~10 M KD (Fig. 1C and 1D). Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/453429v2_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@12a85b4org.highwire.dtl.DTLVardef@aff747org.highwire.dtl.DTLVardef@936401org.highwire.dtl.DTLVardef@8a893b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO p53 binds to CypD at M affinity determined by surface plasmon resonance (SPR). (A) SPR scheme (top) and p53 construct (bottom). (B) SPR sensorgrams of the full length p53 (FLp53)-CypD interaction. The concentrations of FLp53 (from top to bottom) were 2.0, 1.0, 0.5, 0.25, and 0.13 M, respectively. (C) SPR sensorgrams of the NTD-DBD-CypD interaction. The concentrations of NTD-DBD (from top to bottom) were 50, 25, 12.5, 6.25, and 3.13 M, respectively. (D) SPR sensorgrams of the NTD-CypD interaction. The concentrations of NTD (from top to bottom) were 40, 25, 12.5, 6.25, and 3.13 M, respectively. (E) SPR sensorgrams of the interaction between CypD and NTD1-70. The concentrations of NTD1-70 (from top to bottom) were 10, 5, 2.5, 1.25, and 0.63 M, respectively. All SPR sensorgrams are fitted using 1:1 Langmuir binding model (Black curves) in BIAevaluation software 4.0.1. C_FIG

biochemistry↗

bioRxiv2020

Zinc shapes the folding landscape of p53 and establishes a new pathway for reactivating structurally diverse p53 mutants

Missense mutations in the DNA binding domain (DBD) of the p53 tumor suppressor contribute to approximately half of new cancer cases each year worldwide. A primary goal in cancer therapy is to develop drugs that rescue the transcription function of mutant p53. Here we present a thermodynamic model that quantifies and links the major pathways by which mutations inactivate p53. The model is constructed by measuring folding free energies, zinc dissociation constants, and DNA dissociation constants of 20 of the most common DBD mutations in the p53 database. We report here that DBD possesses two unusual properties----one of the highest zinc binding affinities of any eukaryotic protein and extreme instability in the absence of zinc--which are predicted to cause p53 to be poised on the edge of folding/unfolding in the cell, with a major determinant being the concentration of available zinc. Eighty percent of the mutations examined impair either thermodynamic stability, zinc binding affinity, or both. Using a combination of biophysical experiments, cell based assays, and murine cancer models, we demonstrate for the first time that a synthetic zinc metallochaperone not only rescues mutants with decreased zinc affinities, but also mutants that destabilize DBD without impairing zinc binding. The latter is a broad class of p53 mutants of which only one member (Y220C) has been successfully targeted by small molecules. The results suggest that zinc metallochaperones have the capability to treat 120,500 patients per year in the U.S. SUMMARYRestoring tumor suppressing function to mutant p53 has the capability of treating millions of new cancer patients worldwide each year. An important step toward this goal is to categorize the spectrum of mutations based on how they inactivate p53. This study finds that the majority of the most common tumorigenic mutations compromise p53s thermodynamic stability or its interaction with zinc, and demonstrates for the first time that members of both classes can be reactivated in cells by synthetic zinc metallochaperones. These results serve to stratify patients for potential zinc metallochaperone therapy.

biochemistry↗