5.
Structure and channel-forming activityThe three-dimensional structures are available for four Bcl-2 family proteins, two
antiapoptotic members (Bcl-XL and Bcl-2) and two proapoptotic proteins, one from
the multidomain subgroup (Bax) and one from the ‘BH3-domain-only’ group (Bid)
(Muchmore et al., 1996; Chou et al., 1999; McDonnell et al., 1999; Suzuki et al.,
2000; Petros et al., 2001). Surprisingly, the overall structures of all four proteins are
very similar, despite their different activity in the regulation of apoptosis. The proteins
show a fairly compact globular structure with two hydrophobic central helixes (α 5
and α6) surrounded by amphipathic helixes. The helixes are connected by flexible
loop structures, which show a larger variation among the different proteins. In Bid,
the loop between helix 2 and helix 3 contains the caspase-cleavage site. In Bcl-XL and
Bcl-2, the loop N-terminal of the BH3 domain contains several potential
phosphorylation sites. The conserved BH domains, which are involved in protein
interactions and are essential for the activity of the proteins, are all, as expected, located
on the surface of the proteins. However, in Bax, the only protein structure to include
the C-terminal hydrophobic domain, the BH3 cleft, is covered by helix α9 formed
by the hydrophobic domain. Changes in the conformation of the protein might then
regulate its susceptibility for interactions mediated through the BH3 domain and
thus its activity.
The overall structure of the proteins resembles the structure of the pore-forming
domains of some bacterial toxins, diphtheria toxin, and the colicins (Parker and
Pattus, 1993). This suggested that the proteins might possess channel-forming
activity. The surprising finding was that both the pro—and the antiapoptotic proteins
show channel-forming activity in artificial membranes. However, at closer
examination, both the channel-forming conditions and the channel characteristics
were found to differ between the pro—and antiapoptotic members.
The antiapoptotic proteins have channel activity only at low pH, below pH 5.5
(Antonsson et al., 1997; Minn et al., 1997; Schlesinger et al., 1997). This has raised
the question of whether these proteins function as pore-forming proteins under
physiologic conditions, since this is an extreme pH value in physiologic terms.
Although decreases in pH have been reported during apoptosis, these are more
moderate than those that appear to be required for channel formation by the anti-
apoptotic proteins, at least under in vitro conditions. In contrast, the proapoptotic
proteins have channel-forming activity at neutral pH, although the channel activity
is enhanced at lower pH.
Monomeric Bax shows no channel-forming activity. However, after exposure of
the protein to certain detergents, Bax gains channel-forming activity. Channel-
forming properties were shown to be associated with oligomerization of the protein.
Recombinant Bax or cytosolic Bax exposed to Triton X-100 or octyl glucoside forms
oligomers with molecular masses of 80 and 160 kDa respectively (Antonsson et al.,
2000). These would correspond to Bax tetramers and octamers. Oligomerization
appears to take place only when the detergent is present over the critical micellar
MITOCHONDRIA IN APOPTOSIS INDUCTION 127