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. 2000 Oct 24;97(22):11916-21.
doi: 10.1073/pnas.210391597.

Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors

J Parrish  1 , H Metters, L Chen, D Xue
Affiliations

Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors

J Parrish et al. Proc Natl Acad Sci U S A. .

Abstract

Demonstrating in vivo interaction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death in Caenorhabditis elegans. A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4/CED-9 complexes. We show here that an unusual gain-of-function mutation in ced-9 (substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes. Based on a modeled EGL-1/CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4/CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E) in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes.

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Figures

Figure 1
Figure 1
Characterization of interactions between EGL-1 and CED-9 proteins. (A) CED-9(68–251) is sufficient to bind EGL-1. Purified His10CED-9(1–251) or His6CED-9(68–251) (1.5 μg each) was incubated with the indicated amount of EGL-1 and subjected to native PAGE followed by Coomassie blue staining. (B) The G169E substitution in CED-9 impairs the binding of EGL-1 to CED-9. Purified His6CED-9(68–251) or His6CED-9(68–251; G169E) (500 ng each) was incubated with increasing concentrations of purified EGL-1 and then subjected to native PAGE and Western blotting analysis using anti-His6 antibodies. The amounts of bound and unbound CED-9 were quantified using ImageQuant software (Molecular Dynamics). The percentage of CED-9 in complex with EGL-1 is displayed as a function of the amount of EGL-1 used in the binding reactions. (C) Formation of CED-9(G169E)/EGL-1 complexes but not CED-9/EGL-1 complexes is sensitive to the concentration of the non-ionic detergent Nonidet P-40. Binding reactions were carried out as described (B) using 500 ng of CED-9 and EGL-1 proteins in the presence of the indicated amount of Nonidet P-40. (D) Interactions between CED-9 and mutant EGL-1 proteins. Wild-type or mutant EGL-1 proteins (250 ng each) were incubated with 500 ng of the indicated CED-9 proteins in the presence or absence of 0.25% Nonidet P-40, and the assays were carried out as described (B).
Figure 2
Figure 2
Interactions of CED-9 with CED-4 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes. (A) His6CED-9(68–251; G169E) binds GST-CED-4 as well as His6CED-9(68–251). Purified CED-9 proteins (2.5 μg) were incubated with an equivalent amount of GST-CED-4 or GST-Sxl immobilized on glutathione-Sepharose beads. Protein complexes were washed three times with CED-3 buffer (23) containing 0.01% Triton X-100 and subjected to SDS-PAGE followed by Coomassie Blue staining. The CED-9 proteins pulled down by GST-CED-4 are indicated with asterisks (lanes 2–4). (B) CED-4 copurifies with CED-9 proteins in 1:1 ratios. GST-CED-4 and His6CED-9(68–251) or His6CED-9(68–251; G169E) were expressed alone or were coexpressed in E. coli and purified using affinity chromatography. (C) EGL-1 releases CED-4 from CED-4/CED-9 complexes. Five-hundred nanograms of wild-type or mutant EGL-1 was added to approximately 1 μg of purified GST-CED-4/His6CED-9(68–251) or GST-CED-4/His6CED-9(68–251; G169E) complexes immobilized on Ni-NTA beads, and the resulting supernatants were subjected to SDS-PAGE and Western blot analysis using anti-GST antibodies to assess the amount of GST-CED-4 released. As positive controls, 1 M NaCl and 1% Nonidet P-40 were used to disassociate GST-CED-4 from GST-CED-4/CED-9 complexes. (D) EGL-1 displaces CED-4 from CED-4/CED-9 complexes. Five-hundred nanograms of wild-type or mutant EGL-1 was added to approximately 1 μg of purified GST-CED-4/His6CED-9(68–251) or GST-CED-4/His6CED-9(68–251; G169E) complexes immobilized on glutathione-Sepharose beads, and the resulting supernatants were subjected to native PAGE and Western blot analysis using anti-His6 antibodies to visualize the amount of EGL-1/CED-9 complexes released. In lanes 1 and 8, purified CED-9 (250 ng) was loaded as a control for free CED-9 species. EGL-1/CED-9 complexes containing EGL-1(D63R), EGL-1(D64A), or EGL-1(D64G) migrate slower than complexes with wild-type EGL-1 as a result of loss of a negatively charged Asp residue.
Figure 3
Figure 3
Modeled structure of the complex between the BH3 domain of EGL-1 and CED-9. (A) Ribbon stereodrawing of the modeled complex. Backbones of CED-9 and EGL-1 (BH3 domain) are shown in green and magenta, respectively. Potentially critical interface residues are depicted with colored sticks: Y168 (yellow), G169 (red), and R170 (blue) in CED-9 and D63, D64, and D66 (all in red) in EGL-1. The dashed Glu residue at the position of G169 in CED-9 indicates the gain-of-function mutation in CED-9. (B) Sequence alignments between the BH3 domains of EGL-1 and human Bak and between CED-9 and Bcl-xL. The sequence alignment between CED-9 and Bcl-xL was optimized using the SWISS-MODEL program (24). The previously defined seven α-helices (H1–H7) in Bcl-xL are indicated with green bars (25).
Figure 4
Figure 4
EGL-1(D64G) and EGL-1(D64A) induce stronger cell killing in ced-9(n1950) mutants than EGL-1. A 10-kb egl-1 wild-type genomic fragment (hatched box) containing 3.8 kb upstream of the egl-1 start codon and 5.7 kb downstream of the egl-1 stop codon (34) or the corresponding egl-1 genomic fragment carrying either the D64G (gray box) or D64A (empty box) mutation was introduced at 40 μg/ml into ced-1(e1735); egl-1(n1084 n3082) unc-76(e911) or ced-1(e1735); ced-9(n1950) animals with pTG96 (20 μg/ml) (35) and p76–16B (50 μg/ml) (36). The transgenic animals were cultured at three temperatures (15, 20, or 25°C), and the cell-killing activity of the EGL-1 proteins was assessed by counting the number of cell corpses in the head region of 3-fold or later-stage transgenic embryos. All data are averages ± standard deviations of results (n > 50) obtained from three independent transgenic lines.
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