Web Release Date: March 29,
Biophysical Characterization of Recombinant Human Bcl-2 and Its Interactions with
an Inhibitory Ligand, Antimycin A
and
Divisions of Basic Sciences and Clinical Research, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, Washington 98109, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Disease, National Institutes of Health, Bethesda, Maryland 20892, and Department of Protein Biochemistry, ICOS Corporation, 22021 20th Avenue, Southeast, Bothell, Washington 98021
Received October 11, 2000
Revised Manuscript Received January 16, 2001
Abstract:
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-2
22). Characterization of rhBcl-222 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 
-helical protein with unfolding of secondary structure at a Tm of
62.8 
C. Optimal membrane pore formation by rhBcl-222 required negatively charged phospholipids.
The existence of a hydrophobic groove in rhBcl-222 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-222 with a Kd of 0.82 
M. 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.
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