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

Abstract

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