Comparative Biophysical Characterization of p53 with the Pro-apoptotic BAK and the Anti-apoptotic BCL-xL

Begona Sot,Stefan M.V Freund,Alan R Fersht

doi:10.1074/jbc.m705544200

Begona Sot, Stefan M.V Freund + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m705544200

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Abstract

The p53 transcription-independent apoptosis in mitochondria, mediated by its interaction with the pro-apoptotic and the anti-apoptotic members of the Bcl2 family of proteins, has been described in vivo, especially in radiosensitive tissues. We have characterized the interaction of p53 with both the pro-apoptotic Bak and the anti-apoptotic Bcl-x(L) proteins, comparing their affinity and their interaction surfaces, using biophysical techniques such as fluorescence anisotropy, analytical ultracentrifugation, and NMR. We have shown that both proteins interact with only the p53 core domain and not with its N- and C-terminal regions. Further, p53 has a higher affinity for Bcl-x(L) than for Bak, which is consistent with the previously described sequential binding of Bcl-x(L) and Bak by p53. Interestingly, although the interaction with both proteins is electrostatic in character, they have different binding sites. Using NMR spectroscopy, we have determined that Bcl-x(L) interacts with the DNA binding site of p53, but Bak does not interact with this site. A new potential interaction surface for Bak is proposed.

Highlights

The Bcl2 family members regulate the apoptosis mediated by mitochondria
Using NMR spectroscopy, we have determined that Bcl-xL interacts with the DNA binding site of p53, but Bak does not interact with this site
The exact mechanism used by the Bcl2 family to control apoptosis in mitochondria is not completely understood, but it is known that the pro-apoptotic members are active only when they oligomerize

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—Bak⌬C3 (residues 1–190) and BclxL⌬C (residues 1–211) coding sequences, without the C-terminal transmembrane domain, were amplified from a cDNA library (Clontech, multiple tissue cDNA panels) from heart and brain, respectively. The expression vectors for both proteins were made by inserting the coding sequence into the pRSETHisLipoTEV vector [19] using an overlapping PCR strategy [20] Using this strategy, the proteins cleaved from the fusion proteins contained only an additional N-terminal glycine. The proteins were recovered in the flow through, diluted 10 times in 25 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT, 2 mM EDTA, and loaded onto a source 30Q anion exchange column (Amersham Biosciences) equilibrated in the same buffer. The proteins were folded and had characteristic thermal transitions during differential scanning calorimetry: Bcl-xL had a Tm of 76.9 °C and ⌬H ϭ 57 kcal/mol; Bak 72.7 °C and 21 kcal/mol (data not shown). Both proteins were active in the regulation of cytochrome c release from isolated mitochondria.

The abbreviations used are

RESULTS

DISCUSSION