CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

Abstract

The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-offunction CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency

“…Both mutant peptides were able to dissociate the complex as with wild-type EGL-1BH3, but as previously, no dissociation was observed with the BimBH3 peptide. 22 These results demonstrate the exquisite sensitivity of prosurvival protein:BH3 domain interactions and suggest BH3 domain-binding profiles can be readily manipulated.…”

“…21 We have also shown that mammalian BH3-only proteins such as Bim and Bad, unlike EGL-1, cannot dissociate CED-4:CED-9 complexes. 22 This suggests that either these mammalian BH3-only proteins cannot engage CED-9 directly, or alternatively, they bind CED-9 but cannot induce the conformational change necessary for CED-4 release. 11 These studies indicate that the molecular mechanisms, and evolutionary conservation, of nematode and mammalian apoptosis pathways are still not completely understood.…”

“…EGL-1 can only bind some of the mammalian Bcl-2 proteins very weakly in contrast to the much stronger affinity (low nM) for CED-9. 13,14,22 (b) The weak interaction between Bcl-2 and EGL-1 was confirmed by isothermal titration microcalorimetry (ITC) where a K D of 10 mM was determined (left). The control titration of the peptide alone into buffer is also shown (right).…”

“…This high affinity for CED-9 was confirmed by ITC where the EGL-1BH3-C62G peptide bound with a K D of 14 nM (Figure 2d), similar to wild-type EGL-1BH3. 13,22 Finally, as CED-4:CED-9 complex dissociation is dependent upon a conformational change in CED-9 following EGL-1 binding, 13 we also determined the effect of substitution of the cysteine residue in EGL-1BH3 peptide on its ability to dissociate recombinant CED-4:CED-9 complex (Figure 2e). Both mutant peptides were able to dissociate the complex as with wild-type EGL-1BH3, but as previously, no dissociation was observed with the BimBH3 peptide.…”

“…This was confirmed by ITC where no interaction was detected (data not shown), consistent with previous results. 22 To determine that the binding affinity data for the BH3 peptides accurately reflected the ability of full-length BH3-only proteins to interact with pro-survival proteins, we performed co-immunoprecipitation experiments using over-expressed full-length proteins in HEK293T cells ( Figure 1c). As expected, a strong interaction was observed between wild-type EGL-1 and CED-9, but not with Bcl-2 or Bcl-x L , consistent with the low affinity of the EGL-1BH3 peptide measured for these proteins (Figure 1a).…”