Km. Kim et al., Biophysical characterization of recombinant human Bcl-2 and its interactions with an inhibitory ligand, antimycin A, BIOCHEM, 40(16), 2001, pp. 4911-4922
Apoptosis is an essential physiological process, regulated by the family of
Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 re
gulates apoptosis still remains elusive. Here we report the functional stud
ies of recombinant human Bcl-2 with the deletion of 22 residues at the C-te
rminal membrane-anchoring region (rhBcl-2 Delta 22). Characterization of rh
Bcl-2 Delta 22 showed that the recombinant protein is homogeneous and monod
isperse in nondenaturing solutions, stable at room temperature in the prese
nce of a metal chelator, and an or-helical protein with unfolding of second
ary structure at a T, of 62.8 degreesC. Optimal membrane pore formation by
rhBcl-2 Delta 22 required negatively charged phospholipids. The existence o
f a hydrophobic groove in rhBcl-2 Delta 22 was demonstrated by the fluoresc
ence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Ba
k BH3 peptide competed. The respiratory inhibitor antimycin A also bound to
the hydrophobic groove of rhBcl-2 Delta 22 with a K-d of 0.82 muM The opti
mal binding conformation of antimycin A was predicted from molecular dockin
g of antimycin A with the hBcl-2 model created by homology modeling. Antimy
cin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesti
ng that hydrophobic groove-binding compounds may act as selective apoptotic
triggers in tumor cells.