Biophysical characterization of recombinant human Bcl-2 and its interactions with an inhibitory ligand, antimycin A.
Apoptosis is an essential physiological process, regulated by the family of Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 regulates apoptosis still remains elusive. Here we report the functional studies of recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8 degrees C. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. The existence of a hydrophobic groove in rhBcl-2Delta22 was demonstrated by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. The optimal binding conformation of antimycin A was predicted from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.
Kim
K M
KM
Divisions of Basic Sciences and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Giedt
C D
CD
Basañez
G
G
O'Neill
J W
JW
Hill
J J
JJ
Han
Y H
YH
Tzung
S P
SP
Zimmerberg
J
J
Hockenbery
D M
DM
Zhang
K Y
KY
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000378:metabolism; D000968:Antimycin A / Q000737:chemistry* / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002942:Circular Dichroism; D003198:Computer Simulation; D005456:Fluorescent Dyes / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014162:Transfection
D000817:Anilino Naphthalenesulfonates; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D007473:Ion Channels; D008024:Ligands; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D000968:Antimycin A