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

Abstract

Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x L by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim S , which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity. Landmark studies in Caenorhabditis elegans revealed that four genes, CED-9, CED-4, CED-3 and EGL-1, are essential for the programmed cell death (apoptosis) of 131 out of 1090 somatic cells during hermaphrodite nematode development. 1 Of these, EGL-1, CED-3 and CED-4 encode pro-apoptotic proteins whereas CED-9 encodes a pro-survival protein. CED-9 is the nematode homologue of mammalian B-cell lymphoma-2 (Bcl-2) pro-survival proteins (including Bcl-2 itself as well as Bcl-x L , Bcl-w, Mcl-1 and A1), 2 while CED-4 is similar to the mammalian adapter protein APAF-1 3 acting as a positive regulator of caspases, in particular, CED-3. 4 EGL-1 is the nematode Bcl-2 homology domain-3 (BH3)-only protein equivalent to mammalian BH3-only proteins such as Bim, Bad and Noxa. [5][6][7] In nematode cells, CED-9 functions by sequestering CED-4 at the mitochondrial membrane. 8-10 Following a developmental cue, EGL-1 is transcriptionally upregulated and the protein binds CED-9, 5-7 releasing CED-4, allowing its oligomerization. 11 These CED-4 oligomers then translocate to the perinuclear region of the cell 12 where they facilitate CED-3 activation by bringing these caspases into close proximity. 4,9 Biochemical and structural studies have revealed that binding of the EGL-1 BH3 domain into a hydrophobic groove on CED-9 causes a conformational change at the CED-4:CED-9 interface that results in CED-4 dissociation. [13][14][15] The BH3 domains of mammalian BH3-only proteins bind into similar grooves on mammalian pro-survival molecules, but a conformational change as seen in CED-9 has yet to be observed. [16][17][18] The functional equivalence, and hence evolutionary conservation, of CED-9 and mammalian Bcl-2 was demonstrated in st...

Bcl-2 Family Proteins as Therapeutic Targets

“…Small molecules competing with these very tightly bound ligands must have similar levels of activity. In support of this rule, we note that mutated peptides with reduced binding affinities fail to induce apoptosis [55]. Similar results have also been observed with small non-peptidic ligands [56].…”

The role of BH3-only protein Bim extends beyond inhibiting Bcl-2–like prosurvival proteins

“…1 B depicts the Bim mutations generated. Because the binding affinity of a BH3-only protein determines its proapoptotic potential (Chen et al, 2005; Kuwana et al, 2005; Certo et al, 2006; Lee et al, 2008), we determined the affinities of 26-mer peptides spanning the mutant BH3 domains for Bcl-x L and Mcl-1 (Fig. 1 C).…”

“…The affinity of 26-mer BH3 peptides for recombinant murine Bcl-x L ΔC25 and murine Mcl-1ΔN151ΔC23 was determined using a biosensor (model S51; Biacore) as previously described (Lee et al, 2008). The recombinant proteins were expressed and purified as GST fusion proteins using standard techniques.…”

Structural insight into an evolutionarily ancient programmed cell death regulator – the crystal structure of marine sponge BHP2 bound to LB-Bak-2

“…19, 20 Bcl-2 potently protected against apoptosis in the worm, despite its low affinity for pro-apoptotic Egl1 ( K D =7.1  μ M). 21 …”