BclxL changes conformation upon binding to wild-type but not mutant p53 DNA binding domain.
p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-mu, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-mu binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.
Hagn
Franz
F
Department Chemistry, Center for Integrated Protein Science Munich, Technische UniversitätMünchen, 85747 Garching, Germany.
Klein
Christian
C
Demmer
Oliver
O
Marchenko
Natasha
N
Vaseva
Angelina
A
Moll
Ute M
UM
Kessler
Horst
H
D000495:Allosteric Site; D004247:DNA / Q000737:chemistry*; D016158:Genes, p53; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000379:methods; D013449:Sulfonamides / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry*; D051020:bcl-X Protein / Q000737:chemistry*
C545747:2-phenylacetylenesulfonamide; D008024:Ligands; D013449:Sulfonamides; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051020:bcl-X Protein; D004247:DNA