Biophysical characterizations of human mitochondrial transcription factor A and its binding to tumor suppressor p53

Abstract

Human mitochondrial transcription factor A (TFAM) is a multi-functional protein, involved in different aspects of maintaining mitochondrial genome integrity. In this report, we characterized TFAM and its interaction with tumor suppressor p53 using various biophysical methods. DNA-free TFAM is a thermally unstable protein that is in equilibrium between monomers and dimers. Self-association of TFAM is modulated by its basic C-terminal tail. The DNA-binding ability of TFAM is mainly contributed by its first HMG-box, while the second HMG-box has low-DNA-binding capability. We also obtained backbone resonance assignments from the NMR spectra of both HMG-boxes of TFAM. TFAM binds primarily to the N-terminal transactivation domain of p53, with a K(d) of 1.95 +/- 0.19 microM. The C-terminal regulatory domain of p53 provides a secondary binding site for TFAM. The TFAM-p53-binding interface involves both TAD1 and TAD2 sub-domains of p53. Helices alpha1 and alpha2 of the HMG-box constitute the main p53-binding region. Since both TFAM and p53 binds preferentially to distorted DNA, the TFAM-p53 interaction is implicated in DNA damage and repair. In addition, the DNA-binding mechanism of TFAM and biological relevance of the TFAM-p53 interaction are discussed.

Figure 1.

(A) A schematic diagram of various TFAM deletion constructs used in this study. Dashed box indicates region deleted in the shorter TFAM isoform. (B) SDS–PAGE of various TFAM deletion constructs on a NuPAGE® Novex 4–12% Bis–Tris gel.

Figure 2.

(Upper panels) Equilibrium sedimentation AUC of TFAM (5 µM) at speeds of 25 000 r.p.m. (open circle), 30 000 r.p.m. (time) and 35 000 r.p.m (open square). Experiments were conducted at 5°C in 25 mM Tris–HCl pH 7.4, 150 mM NaCl and 1 mM DTT. Data were fitted to either a single-exponential model (A) or a double-exponential model (B). Curve fits were shown as smooth lines. (Lower panels) Residuals of curve fits.

Figure 3.

Binding titration profiles of various TFAM deletion constructs with fluorescein-labeled dsDNA. Experiments were conducted in 25 mM Tris–HCl pH 7.4, 150 mM NaCl and 5 mM DTT. (A) Profiles of HMG1 at three different temperatures, 5°C (diamond), 20°C (triangle) and 37°C (square). (B) Profile of TFAM at 37°C. (C) Profile of TFAM-ΔC at 37°C.

Figure 4.

(A) Proposed DNA-binding mechanism of HMG1. Folded HMG1 exists in equilibrium with unfolded HMG1 and folded HMG1 is competent in DNA binding. The presence of DNA would shift the equilibrium in favor of folded HMG1. (B) Schematic DNA-binding models of TFAM and TFAM-ΔC. The C-tail modulates self-association of TFAM and promotes TFAM dimer formation upon DNA binding. This accounts for more positive co-operativity in DNA binding of TFAM, in comparison to TFAM-ΔC.

Figure 5.

(A) Intrinsic fluorescence emission spectra of TFAM in the absence (red) and presence of 28-bp dsDNA (blue), 28-bp ssDNA (dark yellow) and 50-bp dsDNA (magenta), at excitation wavelength of 280 nm. Experiments were conducted at 25°C in 25 mM Tris–HCl pH 7.4, 150 mM NaCl and 5 mM DTT. Concentrations of both TFAM and DNAs were 500 nM. Control spectra without TFAM were also recorded: buffer (black), 28-bp dsDNA in buffer (green), 28-bp ssDNA in buffer (purple) and 50-bp dsDNA in buffer (cyan). (B) UV CD spectra of 10 µM DNA-free TFAM (continuous line) and TFAM complexed with 4 µM 28-bp dsDNA (broken line), corrected for the background of DNA alone in buffer. Experiments were conducted at 20°C, in 25 mM sodium phosphate pH 7.4, 100 mM KCl and 1 mM 1,4-dithioerythritol.

Figure 6.

Binding titration profiles of HMG1 (diamond), TFAM (triangle) and TFAM-ΔC (square) with AlexaFluor 546-labeled p53N. Experiments were conducted at 37°C, in 25 mM Tris–HCl pH 7.4, 150 mM NaCl and 5 mM DTT.

Figure 7.

2D ¹H,¹⁵N-HSQC NMR spectra of ¹⁵N-labeled p53N (residues 1–93, 100 µM) in the absence (red) and presence of 100 µM of HMG1 (blue). Spectra were recorded at 5°C in 50 mM MES pH 6.8, 100 mM NaCl, 5 mM DTT and 5% (v/v) D₂O.

Figure 8.

Binding titration profiles of TFAM (A) and p53 (B) with fluorescein-labeled linear DNA (circle) or bulged DNA (triangle). Experiments were conducted at 10°C, in 25 mM Tris–HCl pH 7.4, 150 mM NaCl and 5 mM DTT.

Figure 9.

2D ¹H,¹⁵N-HSQC NMR spectra of HMG1 (A) and HMG2 (B), acquired at 15°C and 5°C, respectively. Non-assigned peaks were labeled with NA. Peaks corresponding to side chain amides were not labeled. Both spectra were recorded with 100 µM protein in 25 mM Tris–HCl, 150 mM NaCl, 1 mM DTT and 5% (v/v) D₂O.

Figure 10.

(A) 2D ¹H,¹⁵N-HSQC NMR spectra of ¹³C,¹⁵N-labeled HMG1 (100 µM) in the absence (red) and presence of 100 µM of p53N (blue). (B) 2D ¹H,¹⁵N-HSQC NMR spectra of ¹³C,¹⁵N-labeled HMG1 (100 µM) in the absence (red) and presence of 100 µM of DNA (green). All spectra were recorded at 15°C in 25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM DTT and 5% (v/v) D₂O.

Figure 11.

Comparison of HMG-box domains (HMG1 and HMG2) of TFAM. (A) Stereo views of structural superposition of HMG2 (3fgh, red) overlaid with a bundle of 25 HMG1 models (black). Images were generated using PYMOL. (B) Structure-based sequence alignment of HMG1 and HMG2. Secondary structure elements were shown above the protein sequences. Invariant residues between HMG1 and HMG2 were highlighted in yellow.

Figure 12.

(A) Hydrophobic core of HMG1 model, with Y57, F60, I84, W88, L91, K96 and Y99 indicated. (B) Hydrophobic core of HMG2 (3fgh) with Y162, Y165, V185, W189, L192, K197 and Y200 indicated. Both images were generated using PYMOL.

Figure 13.

Electrostatic surface potential plots of HMG1 model and HMG2 (3fgh), generated with PYMOL. Regions of positive potential were colored in blue, and regions of negative potential were colored in red. (A) Front views showing DNA-binding interfaces. (B) Back views.