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Figure 2. Gossypol induces allosteric Bcl-2 conformational change, which may be responsible for cyto c release. A) Superimposition of 1H-15N HSQC spectra recorded at different Bcl-xL/gossypol ratios: without gossypol (black peaks), 2:1(green peaks), and 1:1(magenta peaks). The residues for which the NH signals showed chemical shift or intensity changes on binding of gossypol are labeled by residue type and number. B) Interaction region between gossypol and Bcl-xL. The dotted blue part is the Bak peptide. All the residues whose NH chemical shifts affected by gossypol titration are indicated in pink. These residues are mostly located at BH1, BH2, and BH3 (also labeled in figure), which form the hydrophobic pocket. C) bak–/–/bax–/– cells were treated with or without gossypol for 24 h, and then immunostained with indicated Bcl-2 Ab, followed by FITC-conjugated secondary Ab, and identified by flow cytometry. Control (purple) and gossypol treated (green). D) bak–/–/bax–/– cells were exposed to 20 µM gossypol for indicated time. Following treatment, cells were lysed in Chaps lysis buffer and immunoprecipitated with the indicated anti-Bcl-2 Ab. Immunoprecipitates were subjected to immunoblotting using anti-Bcl-2 Ab. E) Isolated mitochondria from bak–/–/bax–/– cells were exposed to different concentrations of gossypol as indicated at 25°C for 60 min. After centrifugation, mitochondria were lysed in Chaps buffer and immunoprecipitation was performed as described above.
